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1 Life Science & Biotechnology _Vol.48 Takara Membership Magazine Life Science & Biotechnology Takara Membership Magazine 211 Vol.48 Issues 1. Real Time PCR의개요및주요실험법 2. Cloning의모든것 3. NGS & Microarray Applications 4. Mammalian Inducible Expression 5. Enzyme Immunoassay

2 다카라코리아는 24 시간잠들지않습니다.

3 Life Science & Biotechnology Takara Membership Magazine 211 Vol.48 Real Time PCR 1. Real Time PCR의개요 / Real Time PCR / RT-PCR 2. Real Time PCR 실험 (Step1) / RNA/ 준비 3. Real Time PCR 실험 (Step2) / c 합성 4. Real Time PCR 실험 (Step3) TaqMan Probe를이용한검출 5. Real Time PCR 실험 (Step3) SYBR Green I을이용한검출 6. Real Time PCR 기기 / Thermal Cycler Dice Real Time II Cloning 1. Cloning 실험에대한모든것 2. 제한효소와 Ligation 을이용한 Cloning 3. TA Cloning 4. In-Fusion Cloning 5. In-Fusion Technical Note / 스크리닝용형광바이오센서모형을만드는빠르고효과적인방법 6. 역전사효소와 c 합성 7. Agarose gel 전기영동 8. Technical Note / FlashGel for Recovery Next Generation Sequencing & Microarray Applications 1. Ovation RNA Amplification V2 성능에관한연구 2. WT-Ovation FFPE 을이용한폐조직 FFPE 샘플과신선동결샘플의발현프로파일링비교 3. RNA Amplification : Real Time PCR 이전단계인 RNA 증폭에대한 FAQ staff 발행인 이동근 윤경묵 편집인 황경훈 디자인 유은정 발행처 다카라코리아바이오메디칼 ( 주 ) 주소 서울특별시금천구 가산동 뉴티캐슬 61호 tel / fax / homepage Mammalian Inducible Expression 1. Endless Possibilities / idimerize Inducible Protein Dimerization 2. Inducible Expression / Low Background, High Sensitivity의 3 세대 3. 가장빠르고간편한 Tet Inducible Expression / Tet-Express Inducible Expression Enzyme Immunoassay 1. 골전환율 : 골형성과골흡수의생화학마커 2. Crocodile miniworkstation / 최강의효율과성능, 컴팩트한전자동 ELISA 분석기기 License Notices

4 Real Time PCR 개요 Real Time PCR 의개요 Real Time PCR / RT-PCR Real Time PCR은 PCR 증폭산물을실시간으로모니터링하는해석방법으로, 기존의 PCR법으로는측정하기불가능했던, 정확한정량이가능하다. 또한, PCR법을기본으로하고있기때문에검출감도가높고, mrna 발현해석이나 SNP 타이핑등의유전자해석에필요한필수기술이다. Real Time PCR의용도 Real Time PCR법은유전자발현분석외에 SNP 타이핑, 유전자조작식품 (GMO) 의검사, 바이러스나병원균의검출, 도입유전자의카피수해석등다양한용도로응용되고있다. 또한유전자발현해석과같은정량분석뿐만아니라정성분석에도다양하게활용되고있다. Real Time PCR 반응후에증폭산물을전기영동으로확인할필요가없어, 간편하고신속하게결과를얻을수있을뿐만아니라오염의위험이낮기때문에, 최근에는기존의 PCR법으로검출하던유전자검사를 Real Time PCR로검출하려고하는시도가활발하게진행되고있다. Real Time PCR 정량법의원리 Real Time PCR에서는 PCR 증폭산물을실시간으로모니터링하여타겟유전자의증폭에대한정확한정량이가능하다. 이것이 end-point 에서결과를분석하는일반적인 RT-PCR과크게다른점이다. 아래에정량원리에대해간단하게설명하였다. PCR에서는 1 cycle 마다 가 2배씩기하급수적으로증가해일정이상의 cycle 을반복하면합성된 양은정체기plateau에달한다. 이렇게증폭되는유전자의양을실시간으로모니터한그래프가증폭곡선이다. PCR 증폭산물량이형광검출할수있는양에이르면증폭곡선은급격히상승하기시작하여지수함수적으로시그널이상승한후정체기에이르게된다. a 기기의검출한계 b PCR 증폭량 Fluorescence Detection Concentration Cycle 수 Cycle 수 그림 1. Real Time PCR 의원리 미지의 Sample Copy 수 Threshold Baseline Ct 값Ct 값 기의유전자양이많을수록증폭량은검출가능한역가 threshold 에빠 르게다다르게되므로, 증폭곡선이가파르게상승한다 ( 그림1.a). 따라서, 단계적으로희석한표준샘플serial dilution sample을이용해 Real Time PCR을실시하면, 기유전자양이많은순서대로일정한간격으로나란한증폭곡선을얻을수있다. 여기서, 적당한곳에반응을일으키 c Log concentration 검량선 는최소의역가를설정하면, 반응을일으키는최소의역가와증폭곡선이만나는점인 Ct값 (Threshold Cycle) 을산출할수있다 ( 그림1.b). Ct 값과기주형량의사이에는직선관계가있어그림과같은검량선을작성할수있고, 미지샘플의 Ct값을표준샘플의검량선에적용시키면미지샘플의정량이가능하다 ( 그림1.c). Real Time PCR을위한대표적인형광검출법 Real Time PCR은 PCR 증폭산물의양을형광을이용하여검출한다. 대표적인형광검출방법은 intercalator를이용하는방법과형광 probe 를이용하는방법으로나눌수있다. (1) Intercalator (SYBR Green I) 를이용하는방법대표적인 intercalator로는 SYBR Green I을들수있다. SYBR Green I은 PCR에의해서합성된 double str 에결합해형광을발한다. 이형광량 1) Denaturation Intercalator ( 형광물질 ) 을측정하여 PCR에의해서증폭된산물의양을측정할수있 2) Primer annealing 다. 그림 2. Intercalator를이용한 Real Time PCR의원리 3) Extension (2) Probe를이용한방법 가장대표적인 Probe법은 TaqMan Probe로 TaqMan Probe의 5 end에는검출하고자하는형광물질이, 3 end에는형광을발하지못 하도록제어하는 quencher 가 tag되어있다. TaqMan Probe를이용 한반응에는 1세트의프라이머와하나의 TaqMan Probe를사용한 다. TaqMan Probe는 annealing 단계에서타겟유전자에특이적으 로 hybridization하게되지만, 3 end에있는 quencher 에의해형광 을내지못한다. 다음과정인 extention 단계에서 Real Time PCR시 사용되는 Taq Polymerase의 5 3 exonuclease의활성에의 해, 주형에붙어있 던 TaqMan Probe 1) Denaturation Probe Primer 가깨지면서형광물형광물질 Quencher 질이 quencher로부 터분리되어형광을발할수있게된다. 이때발하는형광량을측정하여타겟유 2) Primer annealing / Probe hybridization Polymerase 3) Extension Hybridize 전자의증폭량을측정할수있다. 그외 probe 방법으로는 Cycling Probe 방법, Molecular Beacon Probe 방법등이있다. 그림 3. TaqMan Probe를이용한 Real Time PCR의원리 2 Life Science & Biotechnology 48

5 TAKARA Real Time PCR Flow Chart Step 1 Template RNA 추출 AGPC 법을이용한 RNA 추출시약 RNAiso Plus (Code 918/919) 2-step Real Time PCR Step 2 Reverse Transcription (2-step Real Time RT-PCR) 15 분만에 Real Time PCR 전용 c 합성가능 Genomic 제거과정을추가한제품도판매 PrimeScript RT Kit Series (Code RR37A/B, RR36A/B, RR47A/B) Step 3 TaqMan Probe 검출용 Real Time PCR Premix Premix Ex Taq (Probe qpcr) (Code RR39A/B) Step 3 SYBR Green I 검출용 Real Time PCR Premix SYBR Premix Ex Taq (Tli RNaseH Plus) (Code RR42A/B) SYBR Premix Ex Taq II (Tli RNaseH Plus) (Code RR82A/B) SYBR Premix DimerEraser (Perfect Real Time) (Code RR91A/B) SYBR Premix Ex Taq GC (Perfect Real Time) (Code RR71A/B) Real Time PCR Primer 디자인 & 합성 Perfect Real Time Support 1-step Real Time PCR Step 2, 3 TaqMan Probe 1-step Real Time PCR One Step PrimeScript RT-PCR Kit (Perfect Real Time) (Code RR64A/B) Step 2, 3 SYBR Green I 1-step Real Time PCR One Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time) (Code RR66A/B) One Step SYBR PrimeScript RT-PCR Kit II (Perfect Real Time) (Code RR86A/B) One Step SYBR PrimeScript PLUS RT-PCR Kit (Perfect Real Time) (Code RR96A/B) Real Time PCR Instrument Thermal Cycler Dice Real Time II (Code TP9/TP96) 3

6 Real Time PCR 실험 Real Time PCR 실험 (Step 1) RNA / 준비 AGPC 법을이용한 RNA 추출시약 RNAiso Plus (Code 918/919) 배양세포, 동식물조직에서효율적으로 total RNA를추출 고효율의 AGPC 법을기반으로한 total RNA 추출시약 샘플량이많은경우나 total RNA를대량으로회수하고자할때최적 유기용매층이선명한붉은색을나타내므로, 상층액과의경계가뚜렷하여, 오염가능성을낮췄다. rm) RNA 표 1. Total RNA 추출 Sample Sample 양추출된 total RNA 양 Mouse liver 1 g 약 5, μg Mouse kidney 1 g 약 3, μg Mouse skeletal muscle 1 g 약 1,5 μg Mouse brain 1 g 약 1,5 μg HL6 cultured cells 1 x 1 7 약 1 μg 담배잎 1 g 약 1, μg 백혈구세포 1 x 1 7 2~4 μg Whole blood* 1 ml 15~2 μg * Whole blood 1 μl에 RNAiso Plus 1 ml 을사용한다. 추출된 RNA 양은샘플의상태와보관상태에따라달라질수있다. 추출 O.K 1 분만에배양세포로부터직접 RT-PCR 용주형 lysate 조제 CellAmp Direct RNA Prep Kit for RT-PCR (Real Time) (Code 3732) 한개의세포로부터도주형 c 증폭가능 CellAmp Whole Transcriptome Amplification Kit (Real Time) Ver.2 (Code 3734) 배양세포로부터간단한조작만으로 Real Time RT-PCR 용주형 lysate 를쉽게조제 소요시간은최단 1분! TAKARA 시약과조합하면약 2시간으로유전자발현해석가능 SYBR Green I, TaqMan probe, 1-step 반응, 2-step 반응에사용가능 1 1,개정도의세포나 2 pg 2 ng의 total RNA로부터효율적으로 c를증폭 미량샘플로부터복수유전자의 Real Time PCR 해석이가능 프로토콜개량으로저발현유전자까지효율적으로증폭가능 Tip! Total RNA 에혼입된게놈 가 PCR 주형으로작용하여 Real Time PCR 결과가부정확할수있다. Total RNA 에혼입된게놈 에대한해결방법은? 게놈 유래의증폭이일어나지않도록프라이머를설계한다. 먼저목적유전자의게놈구조를조사하여사이즈가큰인트론intron을찾는다. 그앞, 뒤엑손exon에서 forward primer와 reverse primer를각각설계한다. 인트론의사이즈가충분히크면게놈유래의증폭이일어나지않고, 인트론의사이즈가작은경우, 게놈유래의증폭이일어나도증폭사이즈가달라융해온도가차이나므로게놈유래의증폭을구별할수있다. 단일엑손유전자나게놈구조가명확하지않다면미리조제한 total RNA를 DNase I 으로처리하여게놈 를제거한다. PrimeScript RT reagent Kit with g Eraser(Code RR47A) 와같은제품을이용하면역전사반응전, 효과적으로 total RNA내의게놈 를제거할수있다. 4 Life Science & Biotechnology 48

7 Real Time PCR 실험 Real Time PCR 실험 (Step 2) c 합성 Real Time RT-PCR 전용 c 합성시약 PrimeScript RT reagent Kit (Perfect Real Time) (Code RR37A/RR37B) Real Time RT-PCR 전용 5x premix c 합성시약 PrimeScript RT Master Mix (Perfect Real Time) (Code RR36A/RR36B) 신장성이뛰어난 PrimeScript RTase로 15분만에반응완료 1 pg~1 μg total RNA를안정적으로역전사가능 (1 μl반응시 ). 폭넓은농도범위에서높은 linearity를얻을수있다. 2 종류의프라이머 (oligo-dt, rom hexamer) 로 Real Time PCR에최적인 c를합성 RR37과 RR36은 oligo-dt와 rom hexamer를동시에이용해대부분의유전자에대해편향성이적게 c를합성한다. Poly A에가까운타겟뿐만아니라 poly A에서멀리떨어진타겟에서도효율적인 Real Time PCR 검출이가능하다. 또한 RR37은 TaqMan Probe 분석에최적화된프로토콜도포함하고있다. 폭넓은범위의농도에서보다정확한 linearity 가능 < 방법 > PrimeScript RT reagent Kit와 A사, B사의 c 합성제품을이용해각각추천조건으로역전사반응을실시한후, Real Time PCR로 stard curve의 linearity를비교하였다. < 결과 > PrimeScript RT reagent Kit를사용했을경우, stard curve가넓은범위에서가장높은 linearity를나타냈다. 당사제품 A사제품 B사제품 역전사반응 (2 μl반응 ) - 주형 : 마우스간 total RNA 2 pg~2 μg, 멸균증류수 Real Time PCR 반응 (25 μl반응 ) - Real Time PCR 반응시약 : SYBR premix Ex Taq II (Perfect Real Time) - 주형 : 위의역전사반응액 2 μl - 타겟 : Rps18 - 프라이머 : Perfect Real Time Support 에서제작한프라이머 - 기기 : Thermal Cycler Dice Real Time RR37 과 RR36 의구별 PrimeScript RT reagent Kit (RR37) - RT 용프라이머선택가능 (oligo-dt 또는 rom hexamer) - Taqman 분석용프로토콜별도표기 PrimeScript RT Master Mix (RR36) - 5X 농도의 premix 타입 (2종류 (oligo-dt, rom hexamer) 의프라이머포함 ) - RNA와 H 2 O만첨가하여 RT 반응수행 Tip! 역전사반응용프라이머의선택가이드 Rom primer : Rom primer를사용하면 mrna 전체에걸쳐효율적으로역전사반응을할수있어 PCR 증폭위치에의존하지않고발현해석을양호하게실시할수있다. 따라서일반적유전자발현해석에는 rom primer가더유리하다. Oligo-dT primer : RNA의 3 말단 poly A부터역전사반응을실시할때, oligo-dt primer를사용한다. 그러나 poly A로부터멀리떨어진경우역전사효율이떨어질수도있기때문에 poly A로부터 1.5kb 이내에서 PCR primer를디자인하여사용하는것이좋다. 또 polya로부터 PCR 증폭위치사이에 2차구조가생기거나 mrna가분해될가능성이있는경우도주의해야한다. Gene specific primer : 특정유전자만을검출하는경우나 1-step RT-PCR을진행하는경우유전자특이적프라이머로역전사반응한다. 이때, PCR용 reverse primer를역전사용프라이머로사용한다. 일반적으로유전자발현해석에서는복수유전자를검출하므로유전자특이적프라이머의사용은적합하지않다. 5

8 Real Time PCR 실험 게놈 제거과정을추가한 Real Time RT-PCR 전용 c 합성시약 PrimeScript RT reagent Kit with g Eraser (Perfect Real Time) (Code RR47A / RR47B) 역전사반응전에주형 RNA 를제품내에포함되어있는 g Eraser 로처리 : 남아있는게놈 를효율적으로제거 게놈 제거 : 42, 2 분, c 합성 : 15 분 게놈 제거효율에따른 Real Time PCR 결과비교 < 방법 > 게놈 (2ng) 가다량으로포함되어있는 total RNA(.2 pg~2 μg ) 를주형으로 PrimeScript RT reagent with g Eraser 와동일한기능의 A사제품을이용해역전사반응후, Real Time PCR 결과를비교하였다. < 결과 > 게놈 가다량으로혼재되어있는 total RNA를주형으로했을경우에도 PrimeScript RT reagent Kit with g Eraser에포함되는 g Eraser로처리하면, 게놈 가완전하게제거됨을확인할수있었다. A 사제품 증폭곡선 증폭곡선 융해곡선 융해곡선 게놈 유래증폭 게놈 유래증폭 샘플 : 마우스심장에서추출한 total RNA (.2 pg, 2 pg, 2 pg, 2 ng, 2 ng, 2 ng, 2 μg ) + 마우스게놈 2 ng 역전사반응 : PrimeScript RT reagent Kit with g Eraser와 A사제품으로각회사에서추천하는프로토콜에따라 c 합성 Real Time PCR 시약 : SYBR Premix Ex Taq (Perfect Real Time) 주형 : 위의역전사반응액 2 μl 타겟 : Rps18 (12bp) 프라이머 : Perfect Real Time Support 에서제작한프라이머 기기 : Thermal Cycler Dice Real Time 각제품의구성물 Primescript RT reagent Kit (Perfect Real Time) (Code RR37A) Primescript RT Master Mix (Perfect Real Time) (Code RR36A) Primescript RT reagent Kit with g Eraser (Perfect Real Time) (Code RR47A) 5 PrimeScript Buffer( for Real Time) *1 4 μl PrimeScript RT Enzyme Mix I *2 1 μl Oligo dt Primer( 5 μm) 1 μl Rom 6 mers( 1 μm) 4 μl RNase Free dh 2 O 1 ml EASY Dilution( for Real Time PCR) 1 ml 5 PrimeScript RT Master Mix (Perfect Real Time) *1 RNase Free dh 2 O EASY Dilution( for Real Time PCR) 4 μl 1 ml 2 1 ml g Eraser 1 μl 5 g Eraser Buffer 2 μl 5 PrimeScript Buffer 2 (for Real Time) *1 4 μl PrimeScript RT Enzyme Mix I *2 1 μl RT Primer Mix *3 4 μl RNase Free dh 2 O 1 ml 2 EASY Dilution (for Real Time PCR) 1 ml *1 :dntp Mixture & Mg 2+ 포함 *2 :RNase Inhibitor 포함 *1 : PrimeScript RTase RNase Inhibitor Oligo dt Primer Rom hexamers dntp Mixture 및반응 buffer ( Mg 2+ ) 포함. *1 :dntp Mixture 포함 *2 :RNase Inhibitor 포함 * 3 : Oligo dt Primer, Rom hexamers 포함 미량샘플로부터의 c 조제키트 Ovation Pico WTA Ovation PicoSL WTA Ovation FFPE WTA WT-Ovation One-Direct NuGEN의핵산조제키트는 SPIA(Single Primer Isormal Amplification) 기술이라고불리는독자적인핵산증폭기술을이용하여, 미량의해석샘플로부터상대적인대표성representation을유지한채핵산을증폭할수있다. 다양한샘플유래의미량 total RNA (1 pg ) c 증폭 Real Time PCR 분석 FFPE, LCM, 병리검체, 단일세포수준, 전혈, FNA 등 Ovation Pico WTA Ovation PicoSL WTA WT-Ovation FFPE WT-Ovation One-Direct Fragmentation Labeling Encore Biotin Module Encore BiotinlL Module Microarray 분석 6 Life Science & Biotechnology 48

9 Real Time PCR 실험 Real Time PCR 실험 (Step 3) TaqMan Probe를 이용한 검출 TaqMan Probe를 이용한 검출: Primer와 Probe 준비 타겟 유전자에 대한 프라이머와 probe를 디자인하여 준비한다. 프라이머와 probe에 의해서 Real Time PCR의 결과가 좌우되므로, 정확한 디자인과 합성이 필요하다 TaqMan Probe 검출용 Real Time PCR 시약 Premix Ex Taq (Probe qpcr) (Code RR39A/RR39B) 높은 효율과 폭넓은 반응 레인지 내열성 RNaseH인 Tli RNaseH를 첨가하여 c 합성 후 남아있는 RNA에 의한 PCR 저해효과를 최대한 억제 Fast PCR에도 적용 가능 : PrimeScript RT Kit과 조합하여 사용 2 premix 시약에 내열성 RNaseH 첨가 (특허 출원중) Fast PCR 가능 보다 정확한 Real Time PCR을 위하여 <Tli RNaseH 첨가> 역전사 반응 종료 Tli RNaseH Plus qpcr reagent 분 분 기존 qpcr reagent 사용 PCR 반응액내 mrna를 분해하여 PCR 효율 증가 반응시간 반응시간 4분 4분 Primer annealing 저해 : PCR 효율 저하 RT 반응후 RNaseH를 별도로 처리할 필요가 없다. PCR 효율 증가, Annealing 효율 증가 = 특별한 과정 불필요 주 형 :: 마 우스 간 total RNA 5 pg~5 ng (일반적으로 qpcr에는 total RNA 1 pg~1 ng 정도의 c 사용) Premix Ex Taq (Probe qpcr) (Code RR39)에는 Tli RNaseH가 2x premix에 포함되어 있어 타겟 : GAPDH (TaqMan Gene Expression Assay 사용) 주형 c 중 잔존 mrna를 분해하기 때문에, 고농도의 c를 주형으로 사용했을 경우라 기 기 : A pplied Biosystem 75 Fast Real-Time 도 잔존 mrna에 의한 PCR 반응 저해가 일어나지 않고, 보다 넓은 다이나믹 레인지에서 정확 한 정량이 가능하다. 각종 Real Time PCR 기기에서 안정된 반응 분 분 PCR 분 분 반응시간 4분 반응시간 4분 반응시간 4분 반응시간 4분 분 분 분 반응시간 4분 반응시간 4분 반응시간 4분 발현분석 : 약 1시간 소요 License Notice [L15] [L52] [M4] [M57] [P7] 7

10 Real Time PCR 실험 1-step Real Time RT-PCR 용시약 (step 2,3) TaqMan probe 를이용한검출 TaqMan probe 검출에최적인 1-step Real Time RT-PCR 용시약 One Step PrimeScript RT-PCR Kit (Perfect Real Time) (Code RR64A/RR64B) 1-step으로조작이간단 샘플수가많을경우나오염을방지하고싶은유전자검사등에특히최적 저발현 mrna의고감도검출에위력발휘 2-step RT-PCR과비교하여 1-step RT-PCR에서사용할수있는 total RNA 양이많고주형의손실도적기때문에, 저발현 mrna의효율적해석이가능. 특정유전자를고감도로검출하는데유리하다. 타사제품보다단시간에해석완료 <RR64> <A사제품 > <B사제품 > 사제품 사제품 A 사제품 B 사제품 분 분 분 분 분 주형타겟기기 마우스간유래 해석시간분분분 각종기기에서 RR39A와 RR64A에대한양호한반응성확인 ABI PRISM 7/77 (Life Technologies) Applied Biosystems 73/75/75 Fast Real-Time PCR 및 StepOnePlus Real-Time PCR (Life Technologies) LightCycler48 (Roche Diagnostics) Thermal Cylcer Dice Real Time II (Takara Bio) Smart Cycler / Smart Cycler II (Cephied) Tip! TaqMan probe 법을이용할때왜 primer 반응성확인이필요한가? TaqMan probe 방법을사용하면비특이적증폭산물은검출되지않지만, 비특이적증폭이일어나기쉬운 primer에서는증폭효율이나검출감도가저하될수있기때문에새롭게디자인하거나합성한 TaqMan probe 반응용 primer에대해서도반응성을확인하는것이좋다. SYBR Green I 법을이용하면 primer 반응을확인할수있다. 8 Life Science & Biotechnology 48 License Notice [L1] [L15] [L52] [M57] [P7]

11 Real Time PCR 실험 Real Time PCR 실험 (Step 3) SYBR Green I 을이용한검출 SYBR Green I 을이용한검출 : Real Time RT-PCR Primer 제작 Perfect Real Time Support - 프라이머검색 : Human, mouse, rat 의 Refseq 등록유전자에대한프라이머서열보유 온라인으로간편하게검색과주문가능 : 주문완료후프라이머서열공개 TAKARA Real Time PCR 시약 SYBR Premix 시리즈 를사용하면반응조건의최적화과정불필요 최고의증폭능력, RNaseH 를이용한정량성향상 SYBR R Premix Ex Taq TM (Tli RNaseH Plus) (Code RR42A/RR42B) 증폭능력, 반응특이성, RNaseH 에의한정량성향상까지최고의밸런스 SYBR R Premix Ex Taq TM II (Tli RNaseH Plus) (Code RR82A/RR82B) 최고의반응특이성 SYBR R Premix DimerEraser R (Perfect Real Time) (Code RR91A/RR91B) GC rich 주형에도적용가능한 Real Time PCR 시약 SYBR R Premix Ex Taq TM GC (Perfect Real Time) (Code R71A/RR71B) SYBR premix 시리즈 (Code RR42/ RR82/ RR91) 의선택가이드 다양한샘플을이용하여 2 step PCR 을하고자할때 : RR42, RR82 비특이적증폭이보임 RR91 사용 : 표준 protocol (3 step PCR) 로반응 고분자에비특이적증폭이보임 RR91 사용 : Option protocol (2 step PCR) 로반응 증폭효율, 범용성 RR42 > RR82 반응특이성 RR42 < RR82 일반적인 Real-Time PCR 적용 비특이적인증폭, primer dimer 를없애고반응특이성을높이고싶을때 Real Time PCR 을통해 유전자발현해석을실시하고있다. g 를주형으로유전자를검출하고싶다. High throughput 해석을실시하고싶다. Cephied 사 Smart Cycle 적용 Perfect Real Time Support 의 Primer 를사용할때 9

12 Real Time PCR 실험 2 premix 시약에내열성 RNaseH 첨가 ( 특허출원중 ) SYBR Premix Ex Taq (Tli RNaseH Plus) (Code RR42) 와 SYBR Premix Ex Taq II (Tli RNaseH Plus) (Code RR82) 에는호열성세균 Thermococcus litoralis 유래의 Tli RNaseH가 2x premix에포함되어있어주형 c 중에남아있는 mrna를분해하기때문에, 고농도의 c를주형으로사용했을경우나타날수있는잔존 mrna에의한 PCR 반응저해없이보다정확한정량이가능하다. RR42 와 RR82 반응성비교 RR42은매우높은증폭효율을나타내며, 타사동등사양의제품에비해비특이적증폭이억제되어보다정확한정량이가능하다. RR82은높은반응특이성을나타내면서, 타사제품에비해반응시간이짧고증폭효율이높다. 타 : PGK1 (GC 54%) 타 : C16orf84 (GC 73%) 비특이적인증폭 특정범위 주형 주형 비특이적인증폭 타 : PLSCR3 (GC 67%) 타 : ATP5F1 (GC 45%) 검량선에서 어 주형주형 주형 Extension time 반응시간 RR82 3 (2 step) 1시간 25분 A사 (2) 6 (2 step) 2시간 1분 C사 3 (3 step) 2시간 13분 게놈 를주형으로사용 :RR71, RR42 및타사제품비교 게놈 를주형으로했을경우, RR71에서는 GC rich target인 CpG isl 영역뿐만아니라일반적인영역에서도타사제품보다증폭효율이높아정확한정량이가능하다. 또한 RR42과비교해서도증폭효율이약간높게나타났다. 사 (2) 사 주형 (pg) 주형 (pg) * 타겟 : CDKN2A-CpG isl 영역 (GC63%) * 타겟 : TFRC (GC38%) License Notice [L11] [L15] [L46] [L52] [M4] [M57] [P5] 1 Life Science & Biotechnology 48

13 Real Time PCR 실험 1-step Real Time RT-PCR 용시약 (Step 2,3) SYBR Green I 을이용한검출 증폭효율을중시한 1-step Real Time RT-PCR 시약 One Step SYBR PrimeScript RT-PCR Kit (Perfect Real Time) (Code RR66A/RR66B) 반응특이성향상, 2 Vials 타입으로조작이간편 One Step SYBR PrimeScript RT-PCR Kit II (Perfect Real Time) (Code RR86A/RR86B) 악세서리단백질의첨가로비특이적증폭을효과적으로억제 One Step SYBR PrimeScript PLUS RT-PCR Kit (Perfect Real Time) (Code RR96A/RR96B) RT 부터 Real Time PCR까지하나의튜브에서연속반응가능하고간편한 1-Step RT-PCR 시약 Premix 시약으로조작이간편하여샘플수가많거나오염을방지해야하는유전자검사등에최적이다. 저발현 mrna의고감도검출에위력발휘! 2-Step RT-PCR에비교하여 1-Step RT-PCR은사용할수있는 total RNA 양이많고주형의손실이적기때문에, 저발현 mrna의해석을효율적으로수행할수있다. 저발현유전자검출에있어서의 1-step 과 2-step 비교 < 방법 > 마우스간유래의 total RNA 2 pg 2 μg에일정량 ( copies) 의 λpolya + RNA-A(Code TX82) 를첨가한것을주형으로서, 1-Step 및 2-Step Real Time RT-PCR을하고, 마우스 Ppia 및 λpolya + RNA-A를검출했다. 한편, 2-Step RT-PCR에서는역전사반응용프라이머로 rom hexamer를사용했다. < 결과 > 저발현유전자인 λpolya + RNA-A의검출은 2-Step RT-PCR에서는 total RNA 2μg를사용했을경우 (B : 청색 ) 에크게반응효율이저하하고, total RNA 2 ng (B : 녹색 ) 에서도반응효율이약간저하되었다. 1-step RT-PCR에서는 total RNA 2μg (D : 청색 ) 에서조금반응효율이저하하고있지만, 2-step RT-PCR에비해적어도 1배의검출감도향상을기대할수있다. 2-Step Real Time RT-PCR 의결과 1-Step Real Time RT-PCR 의결과 A B C D RT 시약 : PrimeScript RT reagent Kit (Perfect Real Time) Real Time PCR 시약 : SYBR Premix Ex Taq II (Perfect Real Time) 시약 : One Step SYBR PrimeScript RT-PCR Kit II (Perfect Real Time) 기기 : Thermal Cycler Dice Real Time A, C : 마우스 Ppia의측정. Total RNA (2 pg 2 μg ) 에내재하는유전자의양을측정해검량선을작성하였다. B, D : λpolya + RNA-A의검출결과. 각 total RNA 에일정량첨가한외부표준 λpolya + RNA-A의측정결과에의해반응효율의차이를확인했다. Tip! 1-Step RT-PCR에서는역전사반응으로유전자특이적프라이머를사용하기때문에 total RNA 양이많아도효율적으로반응할수있다. 반면, 역전사반응에 radom hexamer을사용한 2-Step RT-PCR에서는 total RNA 양이지나치게많으면프라이머가부족하게되어반응효율이저하된다. Oligo dt 프라이머를이용하면 1-Step RT-PCR에가까운결과를얻을수있는경우도있지만, 이경우는 PCR 증폭위치가 3 부근이되도록주의할필요가있다. 범용프라이머를사용하는 2-Step RT-PCR은다양한유전자검출에는대단히편리한방법이나, 특정유전자를고감도로검출하기위해서는 1-Step RT-PCR이유리하다. License Notice [L1] [L11] [L15] [L46] [L52] [M57] [P5] 11

14 Real Time PCR 기기 업그레이드된 Real Time PCR 기기출시 Thermal Cycler Dice Real Time II Thermal Cycler Dice Real Time II (Code TP9) 는 1 Lab 에서 1대씩간편하게이용할수있는새로운 Real Time PCR 장치이다. 96 well plate 타입으로컴팩트하고고성능하드웨어와사용하기편리한소프트웨어를탑재하고있다. 펠티에peltier 소자를이용한안정된온도제어와 well간측정시간차가없는카메라촬영방식으로 well간, 실험간에높은균일성과재현성을구현하고있다. 형광필터는 FAM/SYBR Green I 및 ROX/Texas Red 의 2종류를표준장착하고있다. SYBR Green I검출이나각종 Probe (TaqMan Probe, Cycling Probe) 검출에의한 Real Time PCR이가능하고, FAM 표식 probe 와 ROX 표식 probe에의한 multiplex PCR 도가능하며, 옵션필터 (HEX/VIC, Cy5) 의추가탑재도가능하다. 또, 소프트웨어는편리성을중시하고 MIQE 가이드라인에준한소프트웨어 (Stware Ver. 4) 를장착하고있어셋업부터결과해석까지처음으로조작하는분도간편하게조작이가능하다. 연구자의요구에충실한해석방법을적용하고있어, 클릭하나로고객이원하는방식의다양한해석이가능하다. 고성능하드웨어로정밀한분석가능본 system의가열냉각시스템은 Thermal Cycler Dice 시리즈로이미검증을받은펠티에peltier 소자를사용하여 well간온도차를최소화하였다. 할로겐램프를광원으로사용하며상부에서 well간측정시간차이가없는카메라촬영방식으로형광검출을실행하여 well간검출오차가거의없다. 이결과 well간, 실험간높은균일성과재현성을구현하였다. 높은해석정밀도실현주형을 2배씩희석하여 6단계로반응한결과각농도의주형은정확하게 1 cycle 간격을두고검출됨을확인할수있다. 이것은높은신뢰성으로 2배농도차이도정확하게측정가능하다는것을의미한다, 시약 : SYBR Premix Ex Taq II (Tli RNaseH Plus) (Code RR82A/B) 주형 : 마우스 total RNA 1.562ng~5 ng 상당의 c (N=3) (Actb 유전자 ) (2) SYBR Green I 검출, probe 검출모두가능 2종류의필터 (FAM/SYBR Green I 및 ROX/Texas Red) 를표준장착하고있다. SYBR Green I 검출이나각종 probe(taqman probe, Cycling probe) 검출에의한 Real Time PCR이가능하고, FAM 표식 probe와 ROX 표식 probe에의한 multiplex PCR도가능하다. 옵션필터 (HEX/ VIC, Cy5) 를추가장착하면, 한층더응용의가능성이넓어진다. (3) 컴팩트한사이즈 96well plate type 임에도매우컴팩트한사이즈로기존실험대에설치해쉽게사용할수있다. 편리하고설정이쉬운프로그램 (1) 안정된온도제어와광학시스템이높은해석정밀도실현 96well의반응 검출에서높은균일성을보유하고있어 TaKaRa SYBR Premix Ex Taq II (Tli RNaseH Plus) (Code RR82A/B) 를사용하면 2배차이의해석도가능하다. 96 well 에서높은균일성실현 96 well에동일한반응후 Ct값을조사했다. 모든 well에서균일한반응및검출이되는것을확인할수있다. N=96 의균일성평균 Ct 값 = 22.4 표준편차 =.61 변동계수 (%CV) =.27 시약 : SYBR Premix Ex Taq II (Tli RNaseH Plus) (Code RR82A/B) 주형 : 마우스 c 1ng 소프트웨어는직관적이고심플한 5개의화면으로구성되어있어, setup 부터결과해석까지처음조작하는분도간단하게조작할수있다. Experiment Options NEW Absolute Quantification, Relative Quantification, Plus / Minus Assay, SNP Genotyping Assay 4종류의 Experiment Type을선택할수있으며, 실행종료후 Experiment Type을변경할수있다. Plate Setup Target, Sample 목록관리설정방법을통해체계적인 Plate Setup 이가능하고많은 sample 처리시더욱편리하다. Thermal Prile Setup 조건설정이간단하며, 과거 Run 데이터에서프로그램을간편하게불러올수있다. Result / Analysis 상하로나뉜 2 개의분석화면을독립적으로조작할수있기때문에동일한실험을여러관점에서하나의화면에서분석할수있다. Output Setup NEW 간단한조작으로 RDML 파일을작성하기위한각종텍스트파일을출력할수있다. 12 Life Science & Biotechnology 48

15 Real Time PCR 기기 검량선 상대정량결과 (3) 정성해석 : SNP Genotyping Assay, Plus/Minus Assay [ 추가기능 ] PRTSS(Perfect Real Time Support ) 를사용하는 경우 Primer 정보화일을다운로드하여 Target list로의전환이가능하다. 다양한해석방법대응 (1) Ct 값 (Threshold Cycle) 산출법 [ Scatter Plot ] 측정치의분포를 Scatter Plot로표시합니다. 결과를시각적으로파악하는데최적이다. [ Plate Format ] 각필터의검색결과를종합적으로판단하여자동으로 Plus / Minus 또는 SNP Genotype을판정한다. 컨트롤반응의성공여부와 false positive 가능성도한눈에확인할수있다. [ Crossing Point 법 ] 증폭곡선 (Primary Curve) 와 Threshold 와의교차점에서 Ct 값을산출하는방법이다. Threshold 설정에의해 Ct 값이변화한다. [ 2nd Derivative Maximum 법 ] 증폭속도의변화율이최대치가되는사이클수를 Ct 값으로잡는방법으로검출오차의영향을받지않기때문에형광값의 well 간보정이필요없으며, Crossing Point 처럼 Threshold 설정에따라 Ct 값변동이없기때문에실험간의재현성도높아진다. 법 Internal Control 및 종합판정 타겟판정 법 (2) 정량해석 : 절대정량 상대정량 [ 검량선표시 ] 검량선법은절대정량상대정량모두사용가능하다. 검량선은 PCR 증폭효율과결정계수 ( 상관계수의제곱값 ) 이표시되고분석신뢰성을판단하는지표로유용하다. [ 상대정량결과표시 ] 검량선법또는 Ct( 비교Ct) 법에의해상대정량분석을하고결과는컨트롤샘플을 1로했을때막대그래프로표시된다. 다양한리포트기능출력형식 : Microst Word (.doc), Microst Excel (.xls), Microst PowerPoint (.ppt), PDF (.pdf), 텍스트 (.csv), (.txt), bitmap (.bmp) MIQE 가이드라인준수한최신소프트웨어 (1) RDML 파일출력기능최신 Real Rime PCR 소프트웨어 Thermal Cycler Dice Real Time Stware Ver. 4.에서는 RDML에대응이가능한형식의파일출력이가능하다. 간단조작으로 RDML 파일을작성하기위한각종텍스트파일을출력할수있다 *. * RDML 파일작성에는 RDML Generator 등을이용한다. ugent. be/rdml/choosetool. php 13

16 Real Time PCR 기기 (2) 출력가능한파일종류 Text files (.txt, 8종 ) Data files (1) Quantification file (2) Amplification curve fluorescence data file (3) Melting curve fluorescence data file Annotation files (4) Sample annotation file (5) Target annotation file (6) Run annotation file (7) Thermal cycling condition file (8) Experimenters file Target annotation file은타겟유전자의 GenBank Accession No. 와 primer 서열등의정보를포함하고, Sample annotation file은샘플의명칭이나타입등의정보를포함한다. (3) 상대정량해석소프트웨어 Multiplate RQ 탑재 * Thermal Cycler Dice Real Time II MRQ(Code TP96) 는상대정량해석소프트웨어 Multiplate RQ 를탑재한다파장검출모델이다. Multiplate RQ는 Real Time PCR의반응수증가에대응하기위해개발된소프트웨어로복수의 reference 유전자로보정이가능하여정확한정량이가능하도록개발된상대정량해석소프트웨어다. 복수의 plate 에서실행한결과를불러와상대정량해석을간단히할수있다. Multiplate RQ의특징 복수의 reference 유전자 (housekeeping 유전자등 ) 를이용한보정이가능 복수 plate의실험이필요한다수검사대상, 다수유전자의상대정량을간단하게실시할수있다 복수의 run 파일을한번에정리해석할수있다 검량선법과 Ct법의양쪽모두에대응 그래프표시나여러가지형식으로의출력이가능 * 본소프트웨어는 Thermal Cycler Dice Real Time MRQ (Code TP96) 에만설치되어있다. MIQE 가이드라인이란? 현재 Real Time PCR의데이터기술방법의통일화를목표로국제적으로프로젝트가진행되고있다. MIQE (Minimum Information for Publication Quantitative Real-Time PCR Experiments) 는 Real Time PCR 실험에관한최소한의정보를정하는가이드라인으로, MIBBI 프로젝트 (1) 의일환이다. MIBBI 프로젝트중선행되고있는것은 Micro Array의 MIAME 가이드라인으로서 Micro Array 데이터를논문에투고할때 MIAME 가이드라인에의거하도록권장하고있다. 따라서향후 MIQE 가이드라인에준하여 Real Time PCR 데이터를기술하도록요구될것으로예상된다. (1) Nat. Biotechnol. 28 Aug;26(8): RDML 형식이란? RDML (Real-time PCR Data Markup Language) 은 MIQE 에서정해진정보를기재하기위해서고안된포맷이다 (2). PCR 조건이나 primer 배열등의실험조건이외의 Ct 값이나 Tm 값등의실험데이터도기술할수있다. RDML파일은 XML 형식이므로논문투고에사용할수있으며, 장기적으로연구자간의데이터교환에도활용될것으로기대하고있다. (2) Nucleic Acids Res. 29 Apr;37(7): 표 1. RDML 파일의구성 Thermal Cycler Dice Real Time 시리즈 제품명 Thermal Cycler Dice Thermal Cycler Dice Thermal Cycler Dice Thermal Cycler Dice Real Time II Real Time II MRQ Real Time Single Real Time Single MRQ Code TP9 TP96 TP85 TP87 필 터 FAM TM /SYBR Green l O O O O ROX TM /Texas Red O O - - 추가가능 Option Filter HEX/VIC Cy TM 5 HEX/VIC Cy TM Multiplate RQ (Code TP84) - O - O * 시스템구성 : 본체 + 제어용 PC *1 파장모델인 TP85 과 TP87 은 Option Filter 추가가불가능하다. License Notice [L45] [L47] [P1] 14 Life Science & Biotechnology 48

17 높은형질전환효율과낮은세포독성 Trans IT-22 Transfection Reagent (Code MIR54) Broad Spectrum Delivery - Achieve high expression in many cell types, including hard to transfect cell lines primary cells Outperforms Competitor Reagents - TransIT -22 demonstrated higher protein yield when compared to FuGENE HD, Lipectamine 2, Lipectamine 2 CD Superior Transfection Insect Cells - Obtain higher expression than or insect cell transfection reagents Animal Origin Free - TransIT -22 provides high performance with maximum compatibility Normalized Luciferase Expression (%) Primary MEF K562 Daoy CHO-K1 HEK-293 TransIT -22 FuGENE HD Lipectamine 2 Superior Gene Expression in a Board Spectrum Cell Types using TransIT-22 Transfection Reagent. Cell Types Successfully Transfected at Mirus C6, COS7, CHO-K1, Daoy, DB-TRG-5MG, DI-TNC1, HEK 293, HeLa, HepG2, Jurkat, K562, Neuro-2a, NIH-3T3, Vero, primary HSAEpic, primary MEFs Cell Types Successfully Transfected by Or Researchers Cell lines : 2b4.11, A549, C3H/1T1/2, CHO.K1, HCT116, HEK-293, HEK-293T, HPTCs, INS-1,J774A.1, MCF-7, MDA-MB-231, Monc-1, NCCIT, NIH3T3, NRK52E, NSCLC cell line H522, PC-12, Phoenix Eco, RAW 264.7, RGC-5, Saos-2, SH-5YSY, SV4-Large T antigen rat aortic smooth muscle cells, T-98G, U2OS Primary stem cells : human ES cell derived neural progenitors, human mesenchymal stem cells, mouse ES cell derived cardiomyocytes, primary human airway epilial cells, primary human astrocytes, primary MEFs.

18 Cloning Cloning 실험에대한모든것 Cloning 실험 : Ligation 부터 Directional Cloning 까지 Cloning 의개요 타겟유전자 Target gene를임의의 vector에넣는 cloning 실험은유전자공학실험의기기술중하나이며현재도다양한연구분야에서이용되고있다. 제한효소발견과그응용에의해 cloning은범용성이높은기술로이용되어오고있지만, 근래에는제한효소처리없이진행하는 cloning 방법 (TA cloning, In-Fusion cloning) 의개발에의해한층더편리하게활용할수있게되었다. 또 cloning 실험에서는샘플로부터게놈 혹은 RNA의추출및정제, 역전사반응에의한 c 합성, PCR에의한 단편증폭등을통해 insert 를얻는것도중요한과정이다. 본내용에서는제한효소를이용한전통적인 cloning 방법 (p17), 범용적인 TA cloning 방법 (p19), 최신방법인 In-Fusion cloning(p21) 은물론, cloning 전후에필요한실험과정 (Agarose gel 전기영동 (p26), 정제, c 합성 (p25), PCR, colony PCR 등 ) 도아울러소개하고있다. Cloning 방법 Insert 의말단구조 Vector 준비 제한효소 / Ligation 단편의양끝에제한효소사이트가필요하며, 단편양끝을적절한제한효소로처리한다. 서로다른제한효소를이용하면방향성이있는삽입이가능하다. 처리후, 정제나탈인산화가필요한경우가있다 TA Cloning 3' 말단에 da tailing이가능한 PCR 효소로반응을실시한다. 평활말단증폭산물에는 da를첨가하는반응이필요하다. 삽입방향은결정할수없다. 3' 말단에 dt 가부가되어있는 vector 이용한다. In-Fusion Cloning Vector의말단 15 base와상보적인서열을부가한 PCR primer로 어떤 vector든지사용가능하며 vector를제한효소처리나 타겟유전자를증폭한다. 어떤 vector로도방향성있는삽입이가능하다. PCR을이용해 linear 형태로준비한다. 표 1. 각 Cloning 방법의특징 그림 1. Cloning 실험의기본적인흐름 16 Life Science & Biotechnology 48

19 Cloning 제한효소 / Ligation 을이용한 Cloning 목적 단편을원하는 plasmid vector에삽입하는 cloning 방법은유전자공학실험의가장기본이되는기술이다. 여기에서는제한효소와 T4 ligase( Ligation Kit) 를이용한 cloning 실험을소개하고자한다. [ 실험방법 ] 1. Insert 준비 ( 목적 단편조제 ) (a) 제한효소를이용한 의 cutting 목적 단편의제한효소사이트를확인한후, 사용할 plasmid vector의 cloning 사이트에맞춰사용할제한효소를선정한다. 제한효소 Hind lll 와 BamH I 을이용한 double digestion 의예 1 반응액을조제한다. ( 1 μg ) Hind lll 1 μl BamH I 1 μl 1 x K buffer 2 μl dh 2 O up to 2 μl ( 가볍게탭핑 ) 2 37 에서 1시간반응한다. 3 Agarose gel 전기영동으로확인한다. 반응액의절반정도 (1 μl정도 ) 에 loading buffer를첨가하여전기영동한다. 4 목적 단편이포함된 gel을잘라낸다. 의손상을줄이기위해, UV 노출은최대한줄인다. 5 잘라낸 gel에서 를정제한다 ( 필요시 gel extraction kit 사용 ). 6 정제한 insert 용액을 [A] 로한다 (b) PCR을이용한증폭 Vector와의 ligation에사용하는제한효소사이트를 insert primer의 5' 측에추가 하였다. 이 primer를이용해서 insert 를 PCR 증폭한다. 제한효소사이트 + 5' 에 3 base 이상 nucleotide첨가 TaKaRa Ex Taq Hot Start Version 를사용한예 1 PCR 반응액을조제한후반응한다. 1 Ex Taq Buffer(Mg 2+ plus) 5 μl dntp Mixture( 각 2.5 mm) 4 μl ( 각 2 μm) primer 1.2~1. μm (final conc.) primer 2.2~1. μm (final conc.) <5 ng TaKaRa Ex Taq HS 1.25 U dh 2 O up to 5 μl ( 가볍게탭핑 ) 98 1 sec sec. 3 cycles 72 1 min./kb 2 제한효소를이용하여 단편의양쪽말단을절단한다. ( 필요에따라반응용량증가 ) 1의 PCR 반응액 2 μl Hind lll 1 μl BamH I 1 μl 1 K Buffer 2 μl dh 2 O up to 2μl ( 가볍게탭핑 ) 3 37 에서 1시간반응한다. 4 잘라낸 gel에서 를정제한다 ( 필요시 gel extraction kit 사용 ). 5 정제한 insert 용액을 [A] 로한다 2. Vector plasmid 의준비 1. 목적 plasmid vector (circular) 의 cloning 사이트를제한효소로절단하여벡터를 linear화한다. Plasmid ( 1 μg) Hind lll 1 μl BamH I 1 μl 1 K Buffer 2 μl dh 2 O up to 2 μl ( 가볍게탭핑 ) 2 37 에서 1시간반응한다. 4 반응액으로부터 를정제한다. 5 TE Buffer (2 μl이하 ) 에용해하여 plasmid 용액 [B] 로한다.! 한종류의제한효소로 insert 조제와 plasmid vector를 linear화하는경우에는 linear화한 vector의 self-ligation을방지하기위해아래와같은탈인산화처리한다. 탈인산화처리 (self ligation 방지 ) 제한효소로전단한 plasmid vector Alkaline Phosphatase(BAP) 1 ~ 2 pmol.3 ~.6 U 1 BAP Buffer 5 μl dh 2 O up to 5 μl ( 가볍게탭핑 ) 37~65 에서 3 분간반응 Phenol / Chlororm / Isoamyl alcohol (25 : 24 : 1) 추출 (2 회 ) Chlororm / Isoamyl alcohol (24 : 1) 추출 (1 회 ) 에탄올침전 TE Buffer (2 μl이하 ) 에용해하여 plasmid 용액 [B] 로한다. 5'- 돌출말단의탈인산화는 CIAP(Calf Intestinal Alkaline Phosphatase) 에의한처리로도충분하지만, 평활말단이나 3'- 돌출말단의탈인산화에는 BAP(Bacterial Alkaline Phosphatase) 의사용을권장한다. 17

20 Cloning 3. Insert 와 linear plasmid의 ligation Ligation Kit Mighty Mix 를사용할경우 1 반응액을조제한다. Insert 용액 [A] 25~25 fmol Plasmid 용액 [B] 5 ng(25 fmol) Ligation Mix 7.5 μl dh 2 O up to 15 μl 2 16, 3분반응 ( 또는 25, 5분반응 ) 한다. 4. 형질전환 (Transformation) E. coli HST8 Premium Competent Cells을사용하는경우 1 HST8 competent cell 1 μl를사용직전에얼음위에서융해해안정화시킨후, 14 ml tube로옮긴다 (vortex 불가 ). 2 Ligation 반응용액 1 μl를첨가해얼음에서 3분간방치한다 에서 45간 heat shock 후, 얼음에서 1~2분간방치한다. 4 미리 37 로맞춰놓은 SOC media를최종 1 ml이되도록첨가한다 shaking incubator에서 1시간배양 (16 ~ 225 rpm) 한다. 6 LB plate( 항생제포함 ) 에적당량을 spreading 하고, 37 에서하루동안배양한다. 5. Insert Check PCR 19쪽의 4.Insert Check PCR 참조 6. 배양, Plasmid 정제 19쪽의 5. 배양, Plasmid 정제 참조 26쪽의 Agarose gel 전기영동 참조 27-28쪽의 FlashGel for Recovery 참조! 자주하는질문 Q A Q A Ligation 이나형질전환효율이떨어졌을때에변경할수있는과정은? Ligation 반응시간을늘린다. 용액내에염salt 농도가높으면 ligation 효율이저하된다. 특히에탄올침전시사용되는 ammonium acetate는 ligation시저해작용을하기때문에, 에탄올침전시에염이남지않게깨끗이처리한다. 추출 kit를사용했을경우, 추출액을에탄올로침전하고버퍼를교환하면 ligation 효율을올릴수도있다. 돌출말단cohensive end의 ligation의경우 용액 (vector+insert ) 을 6~65 에서 2~3분정도 incubation 후, 바로차갑게유지한상태에서 Ligation Kit의각시약을첨가해반응을하면, ligation에효율적인돌출말단cohensive end이확보되어형질전환효율이높아질가능성이있다. 위의조작으로 ligation 효율이개선되지않을경우에는 정제를다시하는것을권장한다. 긴단편의 cloning 시유의할점은? 긴단편은 ligation 효율이저하되는경향이있다. TaKaRa Ligation Kit LONG(Code 624) 은긴단편 ligation 에최적화되어있어, 1 kb 이상의 ligation을하는경우에적합하다. 또한 insert 를포함한 plasmid의전체길이가큰경우 ( 특히, 1 kb를넘는경우 ) 에는대장균으로의도입효율이낮아지고, 대장균내에서의 plasmid 의안정성도떨어지므로, 정확한 clone 을확보하기어려워진다. 이러한경우에는큰사이즈의 를효율적으로형질전환할수있는 E.coli HST8 Premium Competent Cells(Code 9128) 의사용을권장한다. [ 관련제품리스트 ] 구분 Code 제품명 용량 16A Hind lll 1, U 제한효소, 탈인산화 11A BamH I 1, U 212A Alkaline Phosphatase (E. coli C75) 5 U 225A Alkaline Phosphatase(Calf intestine) 1, U PCR RR6A TaKaRa Ex Taq Hot Start Version 25 U 623 Ligation Kit Mighty Mix 1 Kit Ligation, 형질전환 624 TaKaRa Ligation Kit LONG 1 Kit 9128 E. coli HST8 Premium Competent Cells 1 Set (1 μl 1) 952 E. coli JM19 Competent Cells 1 Set (1 μl 1) 53 Agarose L3 TAKARA 1 g 전기영동 347A 1 bp Ladder 5 μl ( 1 회 ) 343 λ-hind III digest 1 μg License Notice [L15] [M57] 18 Life Science & Biotechnology 48

21 Cloning TA Cloning Taq Polymerase 와같은 PCR 효소로증폭된 PCR 산물은 3' 말단에 deoxyadenosine(da) 이 1 base 부가된다. TA cloning 은 3' 말단에 deoxythymidine(dt) 1 base를부가한 T-vector와 PCR 증폭산물의 da 1 base가상보적으로결합하는것을이용해, 간편하게 cloning하는방법이다 (insert 5' 말단의인산화는불필요하다 ). [ 실험방법 ] 1. Insert 준비 1 PCR 에의한증폭 (TaKaRa Ex Taq 사용의경우 ) 1 Ex Taq Buffer (Mg 2+ plus) 5 μl dntp Mixture( 각 2.5 mm) 4 μl ( 각 2 μm) primer 1.2~1 μm (final conc.) primer 2.2~1 μm (final conc.) 주형 <5 ng TaKaRa Ex Taq 1.25 U dh 2 O up to 5 μl ( 가볍게탭핑 ) 98 1 sec sec. 3 cycles 72 1 min./kb PrimeSTAR 와같은 α형 PCR 효소는 3' 말단에 da가부가되지않기때문에주의가필요하다, 2 Agarose gel에전기영동하여증폭산물을확인한다. 증폭산물이 single b인경우는다음단계 2. T vector와 ligation 에따라진행한다. Primer dimer나비특이적인 b가확인되었을경우에는전기영동후 agarose gel로부터목적 단편의 b를잘라 를정제한다. 3 잘라낸 gel에서 를정제한다 ( 필요시 gel extraction kit 사용 ). 4 회수한 를 insert 용액으로한다. 2. T-Vector 와 Ligation Mighty TA-cloning Kit 사용할경우 1 새로운 tube 에아래구성물을준비한다. dh 2 O pmd2-t Vector 정제한 PCR산물 (Insert 용액 ) 3 μl 1 μl (5 ng) 1 μl 2 Ligation Mighty Mix를 5 μl첨가하여부드럽게혼합한다 에서 3분간 incubation 한다. 3. 형질전환 (Transformation) E. coli HST8 Premium Competent Cells 사용할경우 1 HST8 competent cell 1 μl를사용직전에얼음위에서융해해안정화시킨후, 14 ml tube로옮긴다 (vortex 불가 ). 2 Ligation 반응용액 1 μl를첨가해얼음속에서 3분간방치한다 에서 45간 heat shock 후, 얼음에서 1~2분간방치한다. 4 미리 37 로맞춰놓은 SOC media를최종 1 ml이되도록첨가한다 shaking incubator에서 1시간배양한다 (16 ~ 225 rpm). 6 LB plate(lb+amp+x-gal) 에적당량을 spreading 한후, 37 에서하루동안배양한다. 7 Blue/White colony 선별을통해 white colony 를선택한다. E.coli JM19를사용하는경우는 Ampicillin, X-Gal, IPTG를첨가한 LB plate에 spreading한다. 37 에서하루동안배양해 Blue/ White colony 선별을통해 white colony 를선택한다. 4. Insert Check PCR EmeraldAmp PCR Master Mix 사용할경우 1 PCR 반응액을준비한다. EmeraldAmp PCR Master Mix(2 Premix) 25 μl Forward Primer.2 μm ( final conc.) Reverse Primer.2 μm ( final conc.) dh 2 O up to 5 μl 2 Plate 상의 colony 를 tip 의앞쪽으로극소량긁어내어 1 의 PCR 반응액에현탁하여증폭한다 98 1 sec sec. 3 cycles 72 1 min./kb 3 PCR 반응액일부를 agarose gel 전기영동으로 insert 를확인한다. 5. 배양, Plasmid 정제 1 목적 plasmid를포함하는 colony 를 2 ml LB(+Amp) 배양액에접종하여하루동안배양한다 2 Plasmid 를정제한다 ( 필요에따라 purification kit를선택하여사용한다 ). 19

22 Cloning! PCR 효소종류와 PCR 산물말단과의관계 PCR Polymerase는크게 2가지종류로나눌수있다. Taq Polymerase로대표되는 Pol I형 (family A) 효소와 Pfu Polymerase로대표되는 α형 (family B) 효소이다. Pol I 형 polymerase와 Pol I 형기반의개량된 PCR 효소 (TaKaRa Ex Taq 등 ) 의증폭산물대부분은 3' 말단에 deoxyadenosine (da) 1 base가부가된다. 이것을이용해 PCR 산물을그대로 TA cloning에사용할수있다. 반면, α형 polymerase를이용한 PCR 증폭산물대부분은효소의강력한 3' 5'exonuclease 활성에의해평활말단이되므로, 그대로는 TA cloning에사용할수없다. 평활말단의 PCR 산물을 TA cloning에이용할경우에는 3' 말단에 da를별도로붙여줘야한다. 다카라에서는 TA cloning용시약 Mighty TA Cloning Kit (Code 628) 와같이, α형 Polymerase로증폭한산물을위한 TA cloning 시약 Mighty TA-cloning Reagent Set for PrimeSTAR(Code 619) 를판매하고있다. 본키트에는평활말단으로되어있는 PCR 증폭산물의 3' 말단에 da를부가하기위한 A-overhang mixture가첨부되어있어간단한과정으로 TA cloning이가능하다. PCR 효소종류 Pol I 형 α형 (family A) (family B) 증폭산물의 da 부가 3 말단 (TA Cloning 가능 ) 평활말단 TaKaRa Ex Taq TaKaRa LA Taq TaKaRa Taq PrimeSTAR 효소명 MightyAmp 시리즈 시리즈 EmeraldAmp SapphireAmp SpeedSTAR! T-Vector에대하여 TA Cloning Kit 외에 2 종류의 T-vector인 T-Vector pmd2 과 T-Vector pmd19(simple) 를별도판매하고있다. 모두형질전환후 Blue/White 선별이가능하다. T-Vector pmd19(simple) 은 puc19에유래하는 multicloning site의모든제한효소사이트를제거하였기때문에, cloning 후 insert 내의제한효소사이트를이용하는경우편리하다. 5kb 이상의 PCR 산물의 Cloning 긴단편의 PCR 산물 (5 kb 이상 ) 의경우는 TA cloning 효율이저하되기때문에, 평활말단 cloning을추천한다. 간편하게말단평활화와인산화를할수있는시약을갖추고있는 Mighty Cloning Reagent Set(Blunt End)(Code 627) 를사용하면편리하다. T T [ 관련제품리스트 ] 구분 Code 제품명 용량 628 Mighty TA-Cloning Kit 2 회 TA Cloning Kit 및 T vector 619 Mighty TA-Cloning Reagent set for PrimeSTAR 2 회 327 T-Vector pmd2 1 μg 3271 T-Vector pmd19 (Simple) 1 μg 9128 E. coli HST8 Premium Competent Cells 1 Set (1 μl 1) 형질전환용 Competent cells 952 E. coli JM19 Competent Cells 1 Set (1 μl 1) 957 E. coli DH5α Competent Cells 1 Set (1 μl 1) Insert Check PCR RR3A EmeraldAmp PCR Master Mix 16 회 53 Agarose L3 TAKARA 1 g 전기영동 3422A 1 bp Ladder (Dye Plus) 5 μl (1 회 ) 343 λ-hind III digest 1 μg 2 Life Science & Biotechnology 48

23 Cloning In-Fusion Cloning Clontech의 In-Fusion Cloning 기술은 In-Fusion 효소를이용해 단편간의말단 15 base의상동서열을 fusion 시켜 cloning 하는기술이다. 사용하는 vector의말단서열을이용하여 cloning 하기때문에모든 vector를사용할수있으며, 추가로어떤서열도첨가되지않는다. 양쪽말단의서열을인식하므로방향성있는 cloning이가능하다. 원리와특징 Insert 종류, cloning 부위, vector에구애받지않는 directional cloning 짧은단편부터긴단편 (5 bp~15 kb) 까지효율적으로 cloning 가능 1회의반응으로여러 단편을동시에 cloning 가능 In-Fusion 반응시간은불과 15분 Linearized Vector 목적 Clone 1 In-Fusion 효소는 단편의말단 15 base의상동서열을이용하여 fusion 시키기때문에 vector의 cloning할위치의양측 15 base와각각상보적인서열을부가한 primer로목적유전자를 PCR 증폭한다. 2 제한효소처리또는 inverse PCR에의해 linear vector를준비하여, 1의 PCR 산물 과 In-Fusion 효소를혼합한다.. 비특이적인증폭이있는경우에는 spin column 정제를, single b의경우는 Cloning Enhancer로처리해서사용한다. 3 In-Fusion 반응 (5, 15 분 ): directional cloning 완료 2 Insert 의 PCR 증폭을위한 In-Fusion Primer 를설계한다 Vector 말단이 5 돌출인경우 Vector 말단이 평활인경우 In-Fusion 효소는각 단편말단 15 base의상동서열을인식해 fusion 시킨다. 그러므로 insert 를 PCR 로증폭할때사용하는 primer 의 5 말단에 linear vector의말단서열과상동의 15 base를추가하는것이핵심이다. 위의그림과같이 linear vector의말단서열에맞춰서 In-Fusion Primer를설계해야한다. 덧붙여 In-Fusion Cloning 반응은 TA Cloning 과달리증폭산물의 A-overhang의유무에의한영향을받지않기때문에, PCR 증폭산물의 A-overhang을고려하지않아도된다. 3 Insert 를 PCR 증폭한다. Advantage HD Polymerase Mix를이용했을경우 5 Advantage HD Buffer(Mg 2+ ) 5 μl dntp Mixture(2.5 mm each) 2 μl FW Primer/RV Primer 각.2~.3 μm Advantage HD Polymerase(2.5 units/ μl ).25 μl 주형 2 ng dh 2 O Vector 말단이 3 돌출인경우 위의염기를 15 개의상동염기서열 로계산합니다. Insert 을증폭하기위해 목적유전자의 primer 서열추가 up to 25 μl [ 실험방법 ] ( 상세한프로토콜은반드시사용설명서를확인해주세요 ) 1. Linear Vector 와 Insert 의조제 1. 사용하는 vector와 cloning 부위를결정해제한효소처리또는 inverse PCR로 vector를선형화한다. : linear vector 용액 [A] 98 1 sec sec. ( 또는 15 sec.) 3 cycles 72 1 min/kb! Primer 디자인온라인사이트 편리한 In-Fusion primer 디자인사이트 (In-Fusion Primer Design Tool) 를이용하십시오. 21

24 Cloning 2. Agarose gel 전기영동으로 PCR 증폭산물확인 1 전기영동의결과에따라서아래와같이 insert 를정제한다 비특이적 PCR 산물이증폭된경우 : - 목적 b 만을잘라 NucleoSpin Extract II 를이용하여 spin column 정제를한다. 정확한단일 b 의 PCR 산물인경우 : - Cloning Enhancer 처리 : PCR 반응액 Cloning Enhancer 37, 15 분 8, 15 분 2 Insert 용액 [B] 로한다 5 μl 2 μl 4. 대장균으로의형질전환 Stellar Competent Cells(Code ) 이나 E. coli HST8 Premium Competent Cells (Code 9128) 등형질전환효율이 cfu/ μg plasmid 이상의 competent cell 사용을추천한다 5. Insert Check PCR, 배양, Plasmid 정제 Insert check PCR 은 19 쪽을참조한다.! 형질전환 colony 수 415 Positive 형질전환체의비율 7/1 3. In-Fusion Cloning 반응 5 In-Fusion HD Enzyme Premix 2 μl Linear Vector [A] x μl 정제 / CE 처리후 PCR 단편 [B] y μl dh 2 O up to 1 μl 5 15 분반응후바로얼음에꽂는다. In-Fusion Cloning이라면 multi-fragments cloning도효율적으로실시할수있다. 각 1 kb의 단편을 In-Fusion HD Cloning Kit를이용해 cloning한후, colony PCR 로 insert를확인하였더니, 1개 clone 중 7개 clone가 positive clone임을확인할수있었다. Code 제품명용량 포함제품 CE *a NS *b Cell *c In-Fusion HD Cloning Kit w/cloning Enhancer 1 rxns In-Fusion HD Cloning Kit w/nucleospin 1 rxns In-Fusion HD Cloning Kit w/competent Cells 1 rxns In-Fusion HD Cloning CE 1 rxns In-Fusion HD Cloning 1 rxns In-Fusion HD Cloning Kit 1 rxns In-Fusion HD EcoDry Cloning Kit 8 rxns Premix 타입의 In-Fusion HD 에는 1rxns 이외에 5 rxns, 1 rxns 제품도있다. In-Fusion HD EcoDry Cloning Kit 는동결건조타입 ( 실온, desiccator 내보존가능 ) 의 In-Fusion HD Kit 이다. Micro-tube 에분주되어있어즉시사용할수있으며, 24 rxns(8 well tube 3 개 ), 96 rxns (96 well plate) 도있다. In-Fusion HD 포함제품 *a Cloning Enhancer (CE) PCR 산물이단일 b인경우에증폭산물을그대로 In-Fusion 반응에이용하기위한전처리시약이다. 이처리를하면 PCR에사용한 primer나주형 plasmid, dntp의영향을받지않고, In-Fusion 효소가최대의성능을나타낼수있다. *b NucleoSpin Extract II (NS) PCR 산물이 multi-b인경우에 gel extraction과 PCR clean-up을동시에할수있는 spin column이다. *c Stellar Competent Cells (Cell) 높은형질전환효율을갖는 competent cell로긴단편 plasmid 형질전환에도높은효율을얻을수있고같은유전자형의다른 competent cell과비교시 colony 형성속도가빠르다. puc계 plasmid의형질전환시에는 β-galactosidase의 α-상보성을이용해 X-Gal을첨가하면재조합체의 Blue/White 선별을할수있다. License Notice [K2] 22 Life Science & Biotechnology 48

25 Cloning Technical Note In-Fusion Cloning 스크리닝용형광바이오센서모형을만드는빠르고효과적인방법 Alix Herr 1, Paul Tewson 2, Anne Marie Quinn 2 & Thomas Hughes 1 1 Department Cell Biology & Neuroscience, Montana State University, Bozeman MT Montana Molecular, 2155 Analysis Drive Suite B, Bozeman, MT 세포내단백질결합가능성을분석하는가장일반적인방법중의하나가 fluorescence resonance energy transfer (FRET) 법이다. FRET법은단백질과연결된두개의리포터 ( 형광물질 ) 사이의결합정도와그양을측정하는것이다. FRET법을기반으로하는바이오센서를만드는데가장어려운점은 donor fluorophore 와 acceptor fluorophore 를세포기능에영향없이단백질이나단백질도메인에결합시키는방법을찾는것이다. 본고에서는 In-Fusion 기술을이용하여수백개의바이오센서모형을신속하고효과적으로제작하는방법에대해기술하고있다. 두개의형광단백질이떨어져있으면 FRET 현상이발생되지않고두형광단백질이너무근접해있어도거리상에작은변화만가능하기때문에 FRET에서도변화가적게발생한다. 바이오센서를만드는과정은수많은모형을만들고테스트하는과정이필연적으로수반된다 (5). 종종링커linker내의한두개의아미노산차이로인해수백종의다양한융합단백질fusion protein이만들어진다. 이모형을만드는일반적인방법은제한효소와라이게이션ligation 방법을이용해클로닝cloning하는것으로많은시간과비용을투자해야만한다. 서론유전적으로형광을발현하는바이오센서를만들어활용하면살아있는세포에서시그널을확인하기쉽다. 현재센서는칼슘calcium (1) 이나대사물질metabolite (2) 그리고 transmembrane voltage (3) 의변화를측정하는데활용되고있다. 일반적으로센서는두개의다른종류의형광단백질을시그널단백질이나단백질도메인에융합시켜제작되며, 단백질의형태가변하면 FRET 효율도변화가나타난다. 이 r 런센서를만드는데가장어려운점은 donor 1. fluorophore와 acceptor fluorophore의적절한위.75 치를찾는것이다. FRET Efficiency Distance (r) 그림 1. FRET depends critically on distance(r) between donor acceptor fluorophores. FRET은두개의형광물질사이에에너지가전이되는것을의미하는데 donor의에너지전이로인해 acceptor 형광단백질의 emission spectrum이변화하게된다 (4). Donor와 acceptor 사이의거리에따라서도에너지효율의차이를보인다 ( 그림 1). 단백질의배위변화 conformational changes는 FRET에서큰효율의차이를만들어내기때문에 FRET 기반의바이오센서를만들때가장중요한것은단백질도메인에 donor 형광단백질과 acceptor 형광단백질을융합시킬최적의위치를찾아내는것이다. In-Fusion 방법을이용하면적은인원으로도신속하고효과적으로수백개의모형을만들수있다. In-Fusion 방법의가장혁신적이고중요한부분은 1회의반응으로 3개의다른 PCR 산물을갖고있는클론을효과적으로제작할수있다는것이다. 단, 각 PCR 산물말단에 15 bp의상동서열homologous sequence 이필요하다. 그러나이것은한정되어있는제한효소사이트를이용하는방법과는다르다. In-Fusion cloning은 3개의 PCR 산물을동시에클로닝가능하므로, 1 회반응으로시그널단백질의다양한위치에 donor 나 acceptor 형광단백질을삽입하는데매우효과적이다. Donor 나 acceptor 형광단백질을포함하는기모듈을만들고난후, In-Fusion을이용하여교차결합하는다른종류의 donor 나 acceptor 형광단백질을제작할수있다. 이방법을활용하면많은종류의구조를손쉽게만들수있다. 이기술은여러가지다른종류의바이오센서프로젝트에활용되고있다. 또한이두과정은매우효과적이고신뢰할만하며자동화를통해쉽게대량으로진행할수있다. 1단계 : In-Fusion 반응으로기모듈제작첫번째단계는가능한구조정보를이용하거나신중한검토를통해시그널단백질활용에최적이라고예상되는위치에 donor 나 acceptor 형광단백질을삽입하는것이다. 시그널단백질과형광단백질을코딩하는부위를 PCR로증폭한후선형화된플라스미드linear plasmid에 In-Fusion 반응으로한개의형광단백질을클로닝한다 ( 그림 2). 이후연계되는반응이이모듈에의해성패가좌우되기때문에 PCR에의한에러는없는지반드시확인해야한다. 23

26 Cloning Donor Modules Acceptor Modules Donor Modules Acceptor Modules 그림 2. Phase I: Four-way In-Fusion reactions produce donor acceptor modules. 그림 4. Combinatorial complexity produces many possible biosensors. 결론 그림 3. Phase II: Three-way In-Fusion reactions cross-couple donor acceptor modules. 2단계 : In-Fusion을이용한 cross-coupling 모듈모형제작 1단계에서만들어진 cross-coupling 모듈제작은자동화할수있다. Acceptor 모듈의 5 말단을증폭할수있는프라이머 1쌍과 donor 모듈의 3 말단을증폭할수있는프라이머 1쌍, 총 2쌍의프라이머가있다면가능하다. 이프라이머를이용해 15 bp의상동서열을갖고있는 2개의단편을증폭하고이를 In-Fusion으로반응하면센서모형이제작된다 ( 그림 3). 이방법을이용하면 donor 모듈의수 (n) 와그에따라결합하게되는 acceptor 모듈의수 (m) 를곱한만큼의다양한 (n x m) 센서모형을만들수있다 ( 그림 4). In-Fusion 기술을이용하면자동분주장치robotic liquid hling와 96-well PCR plate를이용하여대량의실험을쉽게자동화할수있다. PCR 장비에서 In-Fusion 반응을수행한후 transformation 절반을도말한 1 9 CFU cell에서 2개정도의콜로니가나타났다. 에러율은 1% 미만이었고 cross-coupling 반응을위해 2 개의콜로니를선택해서키웠다. In-Fusion Cloning 을이용하면융합단백질과링커사이에 15bp 의상동서열만있다면제한효소사이트를삽입하지않고도수백개의모형도신속하고효과적으로디자인할수있다. 또한효과적이고뛰어난 In- Fusion 반응을이용하면수백개의모형을만드는데전과정을자동화할수있다. References 1. Miyawaki, A., et al. (1999) Proc. Natl. Acad. Sci. USA 96: Looger, L.L., et al. (25) Plant Physiol. 138: Baker, B.J., et al.(28) Brain Cell Biol. 36: Lakowicz, J.R. (26). Principles Fluorescence Spectroscopy (New York: Springer). 5. Shimozono, S. & Miyawaki, A. (28) Methods Cell Biol. 85: Life Science & Biotechnology 48

27 Cloning 역전사효소 & c 합성 정제된 RNA로부터 RT-PCR 또는 library를제작할경우, 우선역전사효소 (Reverse Transcriptate: RTase) 를이용해 c를합성한다. 본고에서는 MMLV유래의 RTase로 1st Str c 합성을위한일반적인방법을소개하고자한다. 현재연구용시약으로사용되고있는역전사효소에는 avian myeloblastosis virus(amv) 에서유래한것과 moloney murineleukemia virus(m- MLV) 에서유래한 2종이있다. 기존에는 mrna 고차구조때문에열안정성이높은 AMV RTase가주로사용되었지만, 최근 MMLV유래의개량형효소 (PrimeScript RTase 등 ) 들의성능이향상되면서 MMLV 유래의역전사효소사용이주류를이루고있다. 또한역전사효소를이용한 c 합성에서는 RNA와 annealing 위치에따라 3종류의 primer를사용할수있다 Oligo dt primer: mrna의 polya tail에 annealing되어, 3 에서부터 c를만든다. 2.. Rom primer( 일반적으로 6nt~ 9nt 정도 ): mrna의모든영역에서 c 합성을시작한다. 3. Specific primer: 목적한특정 c만을합성한다. [ 실험방법 ] 1st str c 합성 PrimeScript Reverse Transcriptase 사용의경우 1 Tube 에주형 RNA/Primer 혼합액을조제한다. Oligo dt primer 5 pmol ( 또는 Rom primer (6 mers) 5 pmol ) ( 또는 Gene specific primer 2 pmol ) dntp mixture(1 mm each) 1 μl Total RNA *1 5 μg ( 또는 mrna 1 μg ) RNase free dh 2 O Up to 1μl 2 65 에서 5 분간 incubation 후, 얼음에서급냉한다. *2 3 아래의반응액을더하여 total 2 μl로한다. 1 반응액 : 주형 RNA/Primer Mixture 1 μl 5X PrimeScript Buffer 4 μl RNase Inhibitor 2 units PrimeScript Reverse Transcriptase 1~2 units RNase free dh 2 O up to 2 μl 4 가볍게 votex한다. 5 아래의조건으로반응한다. Option (3 1 min) *3 42 (~ 5 ) *4 3 ~ 6 min 6 7 에서 15분간 incubation 후얼음에서냉각한다. 위와같은조건으로반응하여생성된 1 st str c는 PCR의주형또는 2 nd str c 합성반응에사용할수있다.! 체크포인트 *1 1 성공적인 c 합성을위해서는순도가높은 RNA 샘플을얻는것이중요하며, 이를위해서세포내에포함되어있는 RNase의작용을억제하는것과사용하는기구나용액등외부에서의 RNase의오염을피해야한다. RNA 조제시실험자의땀이나타액에포함된 RNase의오염을막기위해서작업중불필요하게이야기하지않고, 청결한 disposable 글로브착용을권장한다. *2 1열변성처리에의해 RNA의고차구조를제거한다. *3 1Rom primer 사용시필요하다. *4 1 RNA의분해를방지하기위해서는장시간의고온처리를피하는것이바람직하다. 최근 MMLV 유래개량형 RTase의성능이향상되어 42 에서반응하더라도 RNA 고차구조에의한신장반응저해가나타나지않는 PrimeScript RTase 와같은효소들이많이개발되었다. [ 관련제품리스트 ] 구분 Code 제품명 용량 최고품질의 1st str c의합성 621A PrimeScript II 1st str c Synsis Kit 5 회 범용적인 RT PCR kit RR14A PrimeScript RT-PCR Kit 5 회 RR55A PrimeScript One Step RT-PCR Kit Ver.2 5 회 Full-length c 합성 SMARTer PCR c Synsis Kit 1 회 탁월한신장능력을가진역전사효소 268A PrimeScript Reverse Transcriptase 1, U License Notice [K42] [L1] [L15] [M57] 25

28 Cloning Agarose gel 전기영동 Cloning실험에서 의검출과정량은빼놓을수없는과정이다. 그중에서도 agarose gel 전기영동에의한 검출은가장보편적인방법이다. 본고에서는 agarsoe gel 제작을중심으로소개하고자한다. [ 실험방법 ] 1. Agarose gel 의제작 (1% gel, 15 ml제작 )! 체크포인트 1 Agarose 분말을 1.5 g 덜어낸다. 2 적절한크기의용기에 *1 실온또는냉각한 buffer *2 15ml을흔들어주면서 agarose 분말을넣는다. ( 용기전체의중량을기록해둔다.) 3 랩을덮은후증기가나갈구멍을뚫고, 전자레인지에서가열한다. 때때로전자레인지에서꺼내어가볍게흔들어주기를반복하면서완전하게용해한다 *3 4 가열종료후전자레인지에서꺼내어천천히흔들면서거품을제거한다 ( 필요에따라다시중량을재고, 따뜻한증류수를더해서증발한수분을보충한다 ). 5 실온에서 5 6 까지서서히식힌후, gel tray에용액을붓고 tip 등으로거품제거하고, comb을꽂은후굳힌다. 6 Comb을제거한후전기영동조로옮긴다 ( 바로사용하지않을경우 buffer를채워냉장고에서보관한다 ). 2. 전기영동을위한샘플준비 전기영동을위해샘플용액의 1/1의 1 Loading Buffer를첨가한후잘혼합한다. 분자량마커도이와같이준비한다. Gel의 well에샘플용액을 loading 한다. 3. 전기영동전기영동장치를이용하여전기영동을시작한다. 전류의방향에주의한다 ( 전기영동상류측 (agarose gel well 방향 ) 이음극 (-), 하류측에양극 (+) 을세팅한다 ). 적당하게분리한시점에서전기영동을멈춘다. *1 1 유리삼각플라스크를추천하며, 2~5배큰용량의플라스크를사용하면끓어올라용액이넘치는것을방지할수있다. 3% 이상의고농도 gel을조제할때는한층더큰용기를사용할것을추천한다. *2 13 % 이상의고농도 gel을조제할때 agarose 입자가 buffer에퍼지기어려운경우가있다. 이때미리 buffer를얼음에서 1분정도냉각해두면 agarose 입자가분산하기쉬워진다. *3 1 전자레인지로가열하면용기전체가과열되어격렬하게끓기때문에안전을위해주의가필요하다. 또화상을막기위해서 holder나단열성장갑을사용하는것이바람직하다. Agarose 의종류와농도선택 분리하고싶은범위 Gel 농도 사용하는 agarose 5 bp 미만 3% NuSieve 3:1 등 5 ~ 5, bp 1% Agarose L3, SeaKem LE 등 5, bp 이상.7% < 분자량마커전기영동사진 > 4. Agarose gel 의염색 전기영동후 agarose gel을 EtBr, Gelstar solution(code 5535) SYBR GreenI 등의형광염색액으로염색한다. 1 1 bp Ladder (3% NuSieve 3:1) 2 1 kb Ladder (1% Agarose L3) 3 λ-hind lll digest (1.5% SeaKem GTG) [ 관련제품리스트 ] 구분 Code 제품명 용량 Agarose 53 Agarose L3 TAKARA 1 g 59 NuSieve 3:1 Agarose *1 125 g 54 Seakem LE Agarose *1 5 g 전기영동 buffer 5844 AccuGENE 1X TAE ( Tris-Acetate-EDTA ) Buffer *1 1 L 전기영동장치 AD11 Mupid-2plus *2 1 set 3422A 1 bp Ladder(Dye Plus) 5 μl (1 회 ) 분자량마커 3426A 1 kb Ladder(Dye Plus) 5 μl (1 회 ) 343 λ-hind III digest 1 μg *1 : Lonza 사의제품이다. *2 : Advance 사의제품이다. 26 Life Science & Biotechnology 48

29 Cloning Technical Note FlashGel for Recovery By Mary Riley Hugh White, Lonza Rockl, Inc. 서론 LONZA FlashGel Recovery 을이용하면 agarose gel 만들기와 gel extraction 정제과정없이도 5~1분내 UV에의한손상없이간편하게고효율로 회수가가능하다. Agarose gel 전기영동후 를회수하는방법은기적인분자생물학기술로 b를잘라서스핀컬럼으로 를정제하는방법으로되어있다. B를자를때는 손실을최소로하면서샘플내 agarose양을최소화하도록주의깊게 agarose를제거해야한다. 이과정에서 b를확인하기위해사용하는 UV에의한 손상이일어나지않도록주의해야한다. 컬럼정제과정시 elution buffer 를사용할때 가 membrane에잘 binding하고 elution이잘일어나는지, 그리고 wash step에서는에탄올잔류등에주의해야한다. Agarose gel 전기영동부터 gel 절제하여스핀컬럼정제하기까지최소한시간이상소요된다. LONZA FlashGel Recovery 은 gel을만들어전기영동하는시간과 회수를위해 b를잘라내고 를정제하는과정이모두필요하지않다. FlashGel system은전기영동과 회수까지전과정이 1분이내로가능하며 8% 이상의회수율을보인다. 또한회수된 는전기영동과정제과정시포함될수있는저해물질이나 UV에의한돌연변이발생등의영향없이 증폭이나 cloning, ligation 등에서컬럼으로정제한 와동등이상의효율을보였다. 제품개요 FlashGel 은 5분만에 분리가가능한빠른전기영동속도뿐만아니라 b의전기영동과정을실시간으로확인할수있는광원시스템이결합된혁신적인시스템이다. 또한 FlashGel Recovery 을이용하면 4 1분정도면전기영동하던 gel에서곧바로 를회수할수있다. 이시스템은 b를잘라내고정제하는과정이필요하지않기때문에정제과정상손실을최소화하고 회수율을최대한으로높였다. 출전기분리시간에따라동일한 cassette를이용해회수된샘플을검증할수도있다. 적용방법 FlashGel 이용한회수율 ( 그림 1) 회수율을입증하기위해서, 단계적으로 1/2씩희석serial doubling dilutions ( ng) 한 1, bp Fragments (BioVentures, Inc.) 를 FlashGel Recovery Cassette ( 그림1A: 1 & 2) 로전기영동해서분리한후회수했다. 첫번째단의 well에시료를 loading 한후두번째단의 well의위쪽끝까지전기영동시킨후멈추고두번째단의 well에 FlashGel Recovery Buffer를 2 μl첨가했다. 단편이두번째단의 well로충분히전기영동되도록한다. Pipette으로각 well에서 를회수한다. 회수된시료의 5% 를 1.2% FlashGel Cassette ( 그림1A: 3) 로분석했더니회수범위가 8 9% 정도되었다. 다양한길이의단편도회수가능함을검증하기위해 5 bp - 4, bp의단편을 1 ng씩 FlashGel Recovery Cassette로전기영동한후회수하였다. 회수된시료를 3 μl씩 1.2% FlashGel Cassette 에로딩하여분석하였다 ( 그림 1B). Procedure 1. Load samples in top tier wells. 2. Run until samples reach second tier wells. 3. Stop run add FlashGel Recovery Buffer. 4. Extract from wells by pipette Panel A Concentration Range 1) Pre-Recovery Panel B Size Range Load sample here here Extract here here 가 extract well( 두번째단의 well) 에도착하면 는 agarose gel로부터분리가된상태이기때문에 FlashGel Recovery Buffer를첨가한후 pipette으로쉽게추출해낼수있다. 컴팩트한 FlashGel Dock의빛은 에손상을주거나사람에게해가되지않는다. 거의 8-1% 의높은 회수율뿐만아니라정제과정상저해제가포함될가능성이없으며 PCR이나 cloning과같은다음실험단계에곧바로사용할수있다. FlashGel Cassettes의특허받은염색시료는매우적은양의 도분리와회수가가능하도록할뿐만아니라잠재적돌연변이유발환경에연구자가노출될가능성을최소화했다. 시료추 2) Post-Recovery 3) Recovered Samples 그림 1. Figure 1. recovery recovery capa-capabilitiesbilities. 2 show A: Images FlashGel 1 & 2 show Recovery Cassette before after A: Images 1 & Recovery Cassette before after recovery recovery serial dilutions serial a 1 dilutions bp fragment. a Image 1 bp fragment. Image 3 shows 3 analysis 5% 5% each each recovered recovered sample using a sample using a 1.2% Cassette. 1.2% FlashGel Cassette. B: 1 ng samples fragments B: 1 ng samples fragments ranging in ranging size from in 5 size bp to from 45 bp bp were to recovered, 4 bp were recovered, 3 μl aliquots 3 µl aliquots recovered were were separated on a 1.2% separated on a 1.2% Cassette, FlashGel along with Cassette, QuantLadder along with FlashGel QuantLadder Marker (1 Marker bp to 4 (1 kb). bp to 4 kb). 27

30 Cloning recovery cloning (Fig. 3) Spin column comparison (Fig. 2) Cloning recovered PCR amplicons (Fig. 4) PCR amplification recove 3 ng 1 bp BioMarkers Purified Fragments (BioVentures, Plasmid (pbr322; New Engl BioLabs) was subjected to enzyme digestion was using PstI BamHI using (New control reagents supplied Inc.) were recovered using The Recovery (FG) or restriction A 5 bpdouble amplicon amplified Reagents from GeneA spin column type recovery products (C1 C2) according to Engl BioLabs). Samples restricted were separated inkb Kitusing (Applied Biosystems). An aliquot this were used to amplify 3 manufacturer s instructions. The samples recovered with spin 3.2 GeneAmp fragments weregold recovered column products were separated on a 1% Reliant Gel (Lonza) prior Recovery (FG) or a separated spin column kit (C). 5% recovered each recoveredusing Recovery PCR amplification reaction. reaction was to b excision recovery. Fig. 2 shows comparison 1% sample was analyzed on a 1.2% Cassette 스핀컬럼 정제와 FlashGel 회수 recovered 방법 비교 (그림 2) on a 1.2% Cassette. (Fig. 3A). PCR 단편의 cloning 적용 (그림 separated on. 8recovered μl 후 aliquot Aliquots An 회수 samples were recovered ligated into4) PCR sample was ligated samples separated digested puc19reagents vector using supplied Rapid Time required to complete process from gel electrophoresis to PstI/BamHI bs were recovered. 4 μl into double pjet1.2 using in증폭한 CloneJET PCR Cloning 1,bp Purified Fragments (BioVentures, Inc.) 3ng을 PCR Kit를 이용하여 5 bp 단편을 후, FlashGel Recovery recovered sample was 8 minutes for method, Ligation Kit (Fermentas, Inc.) transformed into NEB 5-alpha on a 1.2% FlashG Kit Inc.). se ligation reactions were n as(fg)과 compared to스핀 9 minutes for Reliant Gelrecovery column systems E. coli Competent Cells (New Engl Aliquots BioLabs).분리한 Thenumber cloning (Fig. 3)(Fermentas, Spin column comparison 2) FlashGel Recovery(Fig. 컬럼 회수 방법(C1과 C2) 으로 전기영동하여 후회수하였다. 회수된 PCR 시료analyzed 8 obtained BioLabs) with both samples were very similar. Plasmid Newtransformed Engl was subjected toe. coli 3 ng 1 bp BioMarkers Purifiedmethods. Fragments (BioVentures, Plasmid (pbr322; colonies recovered fragments w into NEB 5-alpha Competent Cells (New Engl samples from plasmid에 two colonies from라이게이션 each sample were with 에 형질전환 했다. 으로 를 회수했다. 컬럼으로 Reliant Gel double 를 한digested 후 E.coli restriction enzyme digestion using PstI BamHI (New Inc.) were recovered using The스핀 Recovery정제된 시료는 (FG) or 1% amplifications. Fig. 5B show BioLabs). Fig. 4 results colony PCR amplifications run on PstI/BamHI analyzed on ashows 1.2% Cassette along FG C1 C2 Engl BioLabs). 4에서는 restricted werenoseparated spin column type recovery productsgel)로 (C1 전기영동한 C2) according to with a그림 restricted sample vector with insert (Fig. 3B). The중 data무작위로 선택하여 colony PCR (Lonza, premade agarose 후 밴드를 잘라내서 회Samples 형질전환된 콜로니 reactions separated on a Fl several romly selected colonies. The results show amplification clearly were shows appropriate expected insert. manufacturer s instructions. The samples recovered with spin 3.2 kb fragments recoveredexcision using 수했다. 증폭했더니 예측했던 사이즈로 증폭됨을 확인할 cloning 수 있었다. 2. Spin column comparison. that recovered coul products (C). size, indicative successful 이는 column products were separated on a 1% Reliant Gel (Lonza) prior Figure Recovery (FG) or a spin column kit 5%expected each recovered 3 ng 1 bp fragment recovered using Thewas sample on a 1.2% Cassette to b2는 excision recovery. 2 shows comparison 1% (Fig. were recovery cloning 3)Digests Spin1.2% column comparison 2) amplification. original PCR products. 그림 회수된 시료의Fig.1%를 FlashGel Cassette에 전analyzedFlashGel로 회수된 PCR 산물을 곧바로 cloning 실험 사용해 Panel원래의 B Plasmid (Fig. Restriction Recovery (FG) or spin column Panel A Recovered Samples recovery products (C1 C2).(BioVentures, (Fig. 3A). Aliquots recovered samples ligatednew intoengl recovered samples separated on a 1.2% Cassette. type Plasmid were (pbr322; BioLabs) was subjected to 3 ng 1 bp BioMarkers Purified Fragments V Inc.) were recovered using The Recovery (FG) or restriction enzyme double digestion using PstI BamHI (New a 1.2% Cassette, along 부터 회수까지 약 9분 정도 시간이 것에 비해 Kit (Fermentas, Inc.) transformed into NEB 5-alpha recovered sample was 8 minutes for method, Engl BioLabs). Samples restricted were separated spin column type걸렸던 recovery products (C1FlashGel C2) according to withligation QuantLadder Panel A Recovered PCR Products Marker (1 bp to 4 kb). as compared to 9 minutes for Reliant Gel column systemsthee.samples Competent Engl Thefragments number were recovered using manufacturer s recoveredcells with(new spin BioLabs). 3.2 kb 은 전 과정을 수행하는데 8분 정도instructions. 밖에 소요되지coli 않았다. column products were separatedcolonies on a 1% Reliant (Lonza) Recovery (FG)Plasmid or a spin column kit (C). 5% each recovered methods. obtainedgel with both prior samples were very similar. sample was analyzed to b excision recovery. Fig.samples 2 showsfrom comparison 1% from each two colonies sample were digested withon a 1.2% Cassette Time comparison.samples vs. Column Recovery. 3A). Aliquots along recovered samples were ligated into recovered separated onpsti/bamhi a 1.2% Cassette. analyzed on a 1.2% (Fig. Cassette FG C1 C2 PstI/BamHI double Time required to complete process from gel electrophoresis with a restricted sample vectortowith no insert (Fig. 3B).digested The datapuc19 vector using Rapid Ligation Kit (Fermentas, Inc.) transformed into NEB 5-alpha recovered sample was 8 minutesclearly forcolumn shows method, appropriate excision expected insert. Figure 3. Recovery cloning comparison. Samples PstI/BamHI cut pbr322 were Recovery Recovery Method 4. Cloning recovered PCR product. A 5 bp separated 3.2 kbe. fragments were recovered using그림 RecoveryBioLabs). as compared to 9 minutes for Reliant Gel column systems coli Competent Cells (New The number Figure 2. Spin column comparison. FigureEngl 4. Cloning (FG) or spin column kits (C1 C2). A: shows comparison between 5% recovered PCR product. A 5 bp 3 ng 1 bp fragment amplicon was amplified recovered using FlashGel each recovered sample separated on a 1.2% Cassette, along with methods. colonies obtained with both samples were very similar. Plasmid amplicon was amplified recovered using fragments on were Separate recovered using4) The CloningCloning recovered recovered PCR amplicons PCR amplicons (Fig. (Fig. 4) Separate fragments on PCR amplification PCR amplification recovered recovered (Fig. 5). (Fig. 5)8. QuantLadder. B: PstI/BamHI cut plasmid samples from colonies transformed Recovery recovered PCR sample Recovery 8 μl recovered PCR was A Recovered Samples with recovered Panel B Plasmid Digests recovery recovery cloning (Fig. cloning 3) (Fig. 3) Recovery samples; lane V isrestriction digest vector with nrom insert. Or lanes samples from two colonies each sample μl were digested with agarose gel (FG) or spin column Panel contain Reagents QuantLadder GeneAmp Marker (1 bp to 4 kb). A 5 Abp5 amplicon bpengl amplicon was amplified was amplified using control reagents supplied supplied Reagents from from GeneAmp Gold Kit Gold (Applied Kit Biosystems) Biosystems) typebiolabs) recovery products (C1tocontrol C2). sample was ligated into(applied pjet1.2 transformed ligated into vector transformed into E. coli PstI/BamHI on a 1.2% Cassette along PlasmidPlasmid (pbr322; (pbr322; New New Engl BioLabs) was wasusing subjected to 3reagents gments Fragments (BioVentures, (BioVentures, 3 5subjected minutes 6 minutes FG C FG1 FG2 C1 C2analyzed V This image shows FGcomparison C1 C2 1% into E.bp coli Competent Cells. Competent Cells. in in GeneAmp GeneAmp Gold Kit Gold (Applied Kitrecovered (Applied Biosystems). An aliquot An aliquot this this were used were to used amplify amplify 3sample fragments bpinsert fragments in 3B). a multiplex in multiplex with ato restricted vector with no (Fig. Theadata restriction enzyme enzyme double digestion double digestion using using PstI BamHI Biosystems). (New BamHI el veryrecovery (FG) or (FG) or restriction PstI samples separated(new on a 1.2% Cassette, along clearly reaction. shows appropriate excision aliquot expected insert. on next page) BioLabs). BioLabs). Samples Samples separated restricted restricted separated were separated C2) C1 according C2) according to toengl Engl PCR amplification PCR amplification reaction. A 6 μl(continues Aaliquot 6 μl PCR reaction PCR reaction was was reaction reaction was separated was recovered recovered using using Recovery Recovery with were QuantLadder bs from 2. Spin column comparison. Marker (1 bp to 4 kb). ExciseFigure 회수한 를 PCR 증폭의 주형으로 이용 (그림 5)PCR ples overed recovered with with spin spin3.2 kb. 3.2 kb fragments fragments were recovered were recovered using using separated separated on on Recovery Recovery product PCR product. An 8 μlanaliquot 8 μl aliquot recovered recovered PCR sample PCR sample was ligated ligated agarose 3 gel ngwas 1 bp fragment 1% nt Reliant Gel (Lonza) Gel (Lonza) prior Recovery prior 그림 Recovery (FG) or a(fg) spin orcolumn a spin kit column (C). 5% kit (C). each 5% recovered each recovered 5 1were recovered using The Figure 5. PCR amplification reco 2. Spin column comparison. minutes PCR 이용하여 PCR 반응으로 3were bp와were 5 bp bs bs were Kit를 recovered. werea recovered. 4 μl 4multiplex both μl recovered bothpanel recovered PCR fragments PCR fragments into pjet1.2 into pjet1.2 using reagents using reagents supplied supplied in in CloneJET CloneJET PCR Cloning PCR Cloning B Plasmid Restriction Digests Panel Recovered Samples Recovery (FG) or spin column were used to amplify 3 5 b ngsample was analyzed was analyzed onfragment a 1.2% on a 1.2% Cassette Cassette mparison ows comparison 1% 1% sample 3 1 bp type recovery products (C1 C2). analyzed analyzed on a 1.2% on a 1.2% Cassette Cassette (Fig. 5A). (Fig. Aliquots 5A). Aliquots Kit (Fermentas, Kit (Fermentas, Inc.). Aliquots Inc.). Aliquots se ligation se ligation reactions reactions were n were n 6 FG C FG1 FG2 C1 C2 V 단편을 증폭했다. 반응이 끝난 PCR 반응액을 6 씩 FlashGelμl PCR reaction was separa This image shows comparison의 1% (Fig. 3A). samples were ligated were into ligated into hgel 2% Cassette. Cassette. w(fig. ealiquots r e3a). r e caliquots ov e r erecovered du s i nrecovered g T h e samples product bs were recovered. A: 4 recovered samples separated onrecovered Timetransformed comparison. vs. 5-alpha Column Recovery. recovered fragments fragments were also wereused alsoas used templates as templates for 회수하였다. newforpcr new PCR transformed into NEB into NEB 5-alpha E. coli E. Competent coli Competent Cells (New Cells Engl (New Engl PstI/BamHI double digested double digested puc19 vector puc19 using vector using Rapid Rapid selfrom electrophoresis gel electrophoresis to PstI/BamHI to FlashGel Recover Recover Cassette, Recovery (FG) Recovery 으로 전기영동하여 분리한 후 회수된 a 1.2% along fragments were used as templates directly from wells via spin column with QuantLadder Ligationor Ligation Kitspin (Fermentas, KitBioLabs). (Fermentas, Inc.) Inc.) results transformed NEBinto NEB amplifications 5-alpha he Gel method,method, column type recovery amplifications. Fig. 5BFig. shows 5B shows.5 μl.5 aliquots μl aliquots se se amplification amplification se amplification reactions. The re BioLabs). Fig. 4 shows Fig. 4transformed shows into results colony 5-alpha colony PCR PCR amplifications run onrunamplifications. on PCR 4 kb). 단편을 4 씩 로딩하여 1.2% FlashGel Cassette로 분석 1 5 minutes 25 3 minutes Marker (1 bp to analysis on a 1.2% products (C1 (New C2). This imageengl ant column Gel systems column systems E. coli Competent E. coli Competent Cells Cells Engl (New BioLabs). BioLabs). TheThe number The number results amplification Column reactions reactions separated separated on a on a Cassette. Cassette. The results The results show show reactions by gelreactions several several romly romly selected selected colonies. colonies. results The show show amplification 1 & 3 us 3. Recovery cloning comparison. Samples PstI/BamHI cut pbr322 새로운 were comparison samples 1% coloniesshows colonies obtained obtained with both with both samples were very were similar. very similar. PlasmidPlasmid Figure 한were (그림 5A)후 각 단편을 PCR 반응의 주형으로 이용했다. QuantLadder. 그 Recovery Recovery Method Total time: Total time: separated 3.2 kb fragments recovered using Recovery that recovered recovered could be could used besuccessfully used successfully as a template as a template for for reactions 2 & 4 used only single prim products products expected from expected size, indicative size, indicative successful successful cloning cloning that samples on asample 4 1 minutes 1 hour samplesrecovered samples from twrom colonies twoseparated colonies from each each sample were digested were digested with with (FG) or spin column kits (C1 C2). A: shows comparison between 5% Dock Cassettes. 회수된 곧바로 또 다른 PCR 증폭의 주형으로 사 1.2% FlashGel analyzed Cassette, along each recovered sampleamplification. separated림 on a5b에서는 1.2% Cassette,가 along with amplification. original original PCR products. products. PstI/BamHI PstI/BamHI analyzed onpcr a 1.2% on a 1.2% Cassette Cassette along along Separate fragments on Separate fragments on QuantLadder. B: PstI/BamHI cut plasmid samples from colonies transformed comparison. vs. Column Recovery. with FlashGel QuantLadder Time 용되는데 적합함으로 is digest vector with no insert.보여준다. Or lanes with a restricted with a restricted sample sample vectorwith vector no insert with no(fig. insert 3B). (Fig. datathe data with recovered samples; lane V agarose gelthe3b). Summary FlashGel Marker (1 bp to 4 contain QuantLadder Marker (1 bp to 4 kb). clearly shows clearlyappropriate shows appropriate excisionexcision expected 3 expected insert. insert. 3 5 minutes 6 minutes kb). Panel A Panel Recovered A Recovered PCR Products PCR Products Panel B Panel Reamplified B Reamplified PCR Products PCR Products Column Spin gure column 2. Spincomparison. column comparison. Lonza Walkersville C This image shows전기영동 comparison 1% 기영동해서 비교한 결과다. Reliant Gel과 컬럼을 활용하여 도FG 적합함을 의미한다.FG1 FG2 C1 C2 recovered samplesdouble separated on digested puc19 vector using Rapid Time required to complete process from gel electrophoresis to PstI/BamHI Figure 3. Recovery cloning comparison. Samples PstI/BamHI cut pbr322 were Recovery Method separated 3.2 kb fragments using The Recovery were is1recovered a fast effective tool for most Walkersville, MD Recovery (FG) or(continues spin column C2). A: shows comparison between 5% on kits next(c1 page) 3. R e c o v e r y a n d For Research Use Only. Not for use in di each recovered sample separated on a 1.2% Cassette, along with method preparative applications, providing users with an alternative Excise bs from Separate fragments m p a r ifragments s o n. on QuantLadder. B: PstI/BamHI cut plasmid samples from colonies transformed FG1 FG2 C1 FG1C2 FG2 VC1 C2 V on c l o n i n g c oseparate Some components technology th agarose gel Samples PstI/BamHI that both recovery withminimizes with maximizes recovered samples; lane V efficiency is digest vector no insert. Oropportunity lanes agarosecut gel The nucleic acid stain in this product contain QuantLadder Marker (1 bp to 4 kb). pbr322 were separated 5 13 minutes for damage to precious. When used as a combined preparative Probes, Inc., Casset 5 minutes 3 6 minutes is for use only in research applica 3.2 kb fragments analytical tool, Recovery is a highly eco- issued patents. The Dock t Figure 4.Figure Cloning 4. Cloning recovered were recovered PCRrecovered product. PCR product. A 5using bpa 5 bp Reader transilluminator technology a (continues on next page) workflow. ampliconamplicon was amplified was amplified recovered using using nomical method to transform a laboratory recovered FlashGel Recovery 6,914,25. The electrophoresis Recover Recover Recovery Recovery.. 8 μl recovered 8orμl spin recovered PCR PCR Excise bs from (FG) column covered under US Patent 6,95,585. directly from wells via was spin column agarose gel sample sample ligated wasinto ligated kits pjet1.2 into(c1 pjet1.2 transformed transformed C2). A: shows Unless orwise noted, all trademarks into E. coli into Competent E. coli Competent Cells. Cells. 1 5 minutes 25 3 minutes 1 minutes comparison5between The information contained herein is be 5% each recovered scientific technical knowledge Total time: Total time: sample separated on a 1.2% implied, regarding its accuracy or 4 1 minutes 1 hour no warranty is expressed or imp FlashGel Cassette, Figure 3. Recovery Figure 3. Recovery cloning comparison. cloning comparison. Samples Samples PstI/BamHI PstI/BamHI cut pbr322cutwere pbr322 were Figure 5.그림 Figure PCR5.amplification 5. PCR amplification recovered recovered. Reagents. Reagents from from GeneAmp GeneAmp Gold Kit Gold Kit assumes all risks use /or hlin Dock Cassettes. Recover Recover PCR amplification recovered. Reagents from PCR Kit were used to with FlashGel QuantLadder. B: PstI/BamHI cut plasmid samples from colonies were used separatedalong separated 3.2 kb 3.2fragments kb fragments were recovered were recovered using using Recovery Recovery were to amplify used to 3 amplify 3 5 bp fragments 5 bp fragments in a multiplex in a multiplex PCR amplification PCR amplification reaction. reaction. directly from wells via spin column recommendation to infringe any existin (FG) (FG) or spin column or with spin kits column (C1 kitsc2). (C1 A: shows C2). A: shows comparison V comparison between 5% between vector 5% with no insert. transformed recovered samples; lane is digest Or 6 μl amplify 3PCR reaction 5 separated bp was fragments in a multiplex PCR amplification reaction. μl PCR 6 μlpcr reaction was separated on on Recovery Recovery PCR6 PCR each recovered each recovered sample separated sample separated on a 1.2% on a 1.2% Cassette, Cassette, withalong with Copyright 21, Lonza Walkersville, I 1 5along minutes contain FlashGel FlashGel Marker (1 bp to 4 kb).25 3 minutes wasrecovered. separated A: FlashGel Recovery product bs were product reaction product bs were bs were recovered. A:on 4 μl both 4 μl recovered both recovered PCR fragments. PCR fragments. ThePCR recovered The recovered lanes QuantLadder. QuantLadder. B: PstI/BamHI B: PstI/BamHI cut QuantLadder plasmid cutsamples plasmid from samples colonies fromtransformed colonies transformed 8/1 with recovered with recovered samples; lane samples; V islane digest V is vector digest with vector no insert. with no Or insert. lanes Or lanes fragments fragments were used were templates as templates for new for PCRnew amplifications. PCR amplifications..5 μlb:aliquots.5 μl aliquots wereusedwp-fgrecov recovered. A: 4asμlused both recovered PCR fragments. The B: recovered fragments as contain contain QuantLadder QuantLadder Marker (1 Marker bp Total to(1 4 kb). bp to 4 kb). time: Total time:se amplification se amplification reactions. reactions. Theamplifications. resulting The resulting amplification amplification products products were observed were observed for all reactions. for all The templates for new PCR B:.5 μl aliquots se amplification 회수와 cloning 적용 (그림 3) 4 1 minutes 1 hour reactionsresulting reactions by gel analysis by gel analysis on a 1.2% on a 1.2% Cassette, forcassette, along with along by with amplification products were observed all reactions gel analysis on a 1.2% Cassettes. QuantLadder. Reactions 1 & 3 Dock used 1 &3primers used for 3 for 3 5 bp 5 fragments bp fragments while whileprimers Plasmid (pbr322; New Engl BioLabs)를 PstI/BamHI 으 QuantLadder. FlashGel Reactions Cassette, along with primers FlashGel QuantLadder. Reactions 1 & 3 used reactionsreactions 2 & 4 used 2 &only 4 used single onlyprimer singleset. primer set. (continues(continues on next page) on next page) for 3 5 bp fragments while reactions 2 & 4 used only single primer set. 1 ng bp 1 fragment bp fragment ered ere recovered using Theusing The Panel A Recovered Panel A Recovered Samples Samples ystem ecovery (FG) or spin (FG) column or spin column ype ery products recovery (C1 products C2). (C1 C2). FG C FG C his shows image comparison shows comparison 1% 1% ered recovered samples separated samples separated on on hgel 1.2% Cassette, along Cassette, along ashgel ith QuantLadder QuantLadder NA ashgel Marker (1 Marker bp to(1 4 kb).bp to 4 kb). ecovery. olumn Column eryrecovery Method Method Separate fragmentsfragments on on arose gel agarose gel 6 minutes 3 6 minutes bs Excise from bs from arose gel agarose gel minutes 5 1 minutes over Recover pin column via spin column 3 minutes 25 3 minutes al time: Total time: 1 hour 1 hour Recovery 그림 Panel B Plasmid Panel B Restriction Plasmid Restriction Digests Digests 로 이중으로 절단한 후, FlashGel Recovery (FG)과 스핀 컬 License Notice [FlashGel] 럼 정제 키트 (C)를 이용해 3.2 kb 단편을 전기영동한 후 회수 Summary Summary 하였다. 각 방법으로 회수된 시료의 5%를 1.2% FlashGel Lonza Walkersville Lonza Walkersville Cassette (그림 3A)로 전기영동하여 분석했다. 또한 회수된 를 Walkersville, Walkersville, MD 21793MD The The Recovery Recovery is a fast is a fast effective effective tool fortool most for most For Research For Research Use Only.Use NotOnly. for use Notinfor diagnostic use in diagnostic procedures. procedures. Pstpreparative I/Bam HI으로 절단된 puc19 vector에 라이게이션 한후 E. coli preparative applications, applications, providing providing users with users anwith alternative an alternative method method components Some components technology technology are sold under are sold licensing under licensing agreements. agreements. both thatmaximizes both maximizes recovery recovery efficiency efficiency minimizes minimizes opportunity opportunity 에 that 형질전환 했다. 두 방법으로 회수된 에서 나온 콜로니 수는 매 Some The nucleic Theacid nucleic stainacid in this stainproduct in this is product manufactured is manufactured sold under sold license underfrom license Molecular from Molecular Probes, Inc., Probes, Inc., CassetteCassette is sold under is sold license underfrom license Invitrogen from Invitrogen IP Holdings, IP Holdings, Inc, Inc, damage for damage to precious to precious. When. used When as used a combined as a선택해 combined preparative preparative 우 for 비슷했다. 각 시료의 형질전환된 콜로니 2개씩 plasmid를 is for useisonly for use in research only in research applications applications or qualityorcontrol, quality control, is covered is by covered pending by pending analytical analytical tool, tool, Recovery Recovery is a highly is a highly eco- ecoreader Reader transilluminator transilluminator technology technology is covered is under covered USunder Patents US 6,198,17; Patents 6,198,17; 6,512,236; 6,512,236; nomical nomical method method to transform to transform a laboratory a laboratory workflow. workflow. issued patents. The The Dock technology Dock technology contains contains Clare Chemical Clare Chemical Research,Research, Inc. DarkInc. Dark 추출한 후 Pst I/BamHI으로 절단한 후 전기영동했다(그림 3B). 전기 issued patents. 6,914,25. The electrophoresis The electrophoresis technology technology is licensed is licensed from Temple from University Temple University is is 영동 결과는 모두 정확하게 vector와 insert로 절단된 것을 확인할 수 6,914,25. Dock Cassettes. Dock Cassettes. 있었다 Life Science & Biotechnology 48 covered under covered USunder PatentUS6,95,585. Patent 6,95,585. Unless orwise Unless orwise noted, allnoted, trademarks all trademarks herein areherein marksare marks Lonza Group Lonza or its Group affiliates. or its affiliates. The information The information containedcontained herein is herein believed is believed to be correct to be correct corresponds corresponds to latest to state latest state scientific scientific technical technical knowledge. knowledge. However,However, no warranty no warranty is made,iseir made,expressed eir expressed or or implied, regarding implied, regarding its accuracy its accuracy or results or toresults be obtained to be obtained from from use such useinformation such information no warranty no warranty is expressed is expressed or implied or concerning implied concerning use se use products. se products. The buyer The buyer assumesassumes all risks alluse risks /or usehling. /or hling. No statement No statement is intended is intended or shouldorbeshould construed be construed as a as a recommendation recommendation to infringe toany infringe existing anypatent. existing patent. Copyright Copyright 21, Lonza 21, Walkersville, Lonza Walkersville, Inc. All rights Inc. All reserved. rights reserved. WP-FGRecov WP-FGRecov 8/1 8/1 MB-WP1 MB-WP1

31 nuview Tris-Glycine Precast Gels 2 분후마법처럼 B 가나타납니다. Fast Run Times The formulation nuvie developed in 125 Vh as c Laemmli gels same Vh when developed in Tri time to run gels. Protein 전기영동할때아직도 Color Protein Marker에모든것을의존하시나요? Marker는이제 Size 확인에만사용하세요. 내샘플의전기영동상태를눈으로직접확인할수있습니다. 별도의염색없이바로 b 확인가능 : 일반 Transilluminator에 2분 긴보관기간 : 4 에서 18개월 ( 실온에서 6개월 ) 3분이면전기영동완료 : high voltage 전기영동가능 다양한전기영동장치에적용가능 : 다양한 size의 gel 보유, 현재사용하고있는전기영동장치에바로적용 다양한전기영동 buffer 사용가능 : 목적에따라 MES, MOPS, Glycine buffer 사용가능 Run Run samples in in your tank tank Run Confirm Transfer Efficiency nuview Cassette Selection NN (fits XCell SureLock tanks) NB (fits Mini- Protean tanks) minutes View View Stability & Easy St 18 month storage life (4 manufacture or 6 months (25 C) R 25 NG (fits all or tanks) 3 After After run, visualize your your gel gel UV under UV Note: Stard Laemmli gel run in Tris-G At 4 After After transfer, visualize residual After After transfer, visualize your your protein in UV protein in gel under UV PVDF under UV PVDF membrane under UV 2 minute visualization under UV light NuSep nuview Precast Gel 을이용한 Protein 전기영동 At 37 NuSep Invitrogen Bio-Rad nuview NB Casstte nuview NN Casstte nuview NG Casstte NuPAGE Bis-Tris Mini-PROTEAN TGX UV B Visualization in minutes x x 2 minutes 2 minutes 2 minutes 16 hours (staining) 16 hours (staining) Recommended Running Buffer Tris-Glycine, Tris-MES, Tris-MOPS Tris-Glycine, Tris-MES, Tris-MOPS Tris-Glycine Compatible Tank Mini-PROTEAN Tanks Xcell SureLock Tanks All or 1cm Tanks Xcell SureLock Tanks Mini-PROTEAN Tanks Run Tims 55 minutes ㅡ 35 minutes (Tris-Glycine) (35 minutes-mes Buffer) 3 minutes (35 minutes-mes Buffer) 3 minutes Recommended 25 V 25 V 25 V 2 V 2 V Voltag e SDS in Gel x x x x x Shelf life at 4 18 month 18 month 18 month 12 month 12 month Casstte Size Wide 1.cm 1.cm 1.cm 1.cm 1.cm High 8.5cm 1.cm 8.cm 1.cm 8.cm Thick.5cm.7cm.5cm.7cm.46cm Gel Size Wide 8.cm 8.cm 8.cm 8.cm 8.6cm High 7.3cm 8.8cm 6.8cm 7.5cm 7.3cm Thick.1cm.1cm.1cm.1cm.1cm

32 NGS Microarray Applications Technical Note 1 Ovation RNA Amplification V2 성능에관한연구 서론 재료및방법 NuGEN의 RNA 증폭제품시리즈를이용하면매우적은양의 RNA 를사용하여탁월한유전자발현결과를얻을수있다. 많은연구에 Ovation Biotin RNA Amplification labeling (Code 23) 제품이사용되었으며, 이제품은 Affymetrix GeneChip arrays에적용하기위한증폭amplification, 단편화fragmentation, 라벨링labeling 과정에필요한구성품을모두포함하고있어 microarray 분석을위한 c 증폭및 array 결과검증을위한 qpcr 분석에사용되고있다. NuGEN은결과의일관성, 실험시간과비용감소등의장점을갖고있는다양한제품을갖추고있다. 이들제품중 Ovation RNA Amplification V2 (Code 31-12, 31-6) 를사용하여증폭한 c는바로 qpcr에사용할수있으며또한이후에 FL-Ovation c Biotin Module V2 (Code 42-12, 42-6) 를사용하여 c를단편화와라벨링 (F&L) 하면 GeneChip array 분석에사용할수도있다. F&L 모듈은간단하고최적화된방법으로 2시간이내에타겟을준비할수있으며별도의정제단계가필요없기때문에보다쉽고, 보다샘플준비의자동화가용이하며, 비용, 처리시간및에러율까지줄일수있다. Nugen의제품라인은연구자가최대한다양하게선택할수있도록모듈식으로구성되어있다. 그예로, 연구자가처음에는샘플을이용하여 qpcr만을수행하지만, 후에동일한샘플의 c를이용하여 microarray에적용하고자한다면, F&L 사용이가능하도록 c를증폭하면서당장은기실험에필요한 c를증폭제품만을구입할수있다. 이러한 NuGEN의제품라인은대형공동프로젝트에사용하면상당히유용할것이다. 왜냐하면다양한분야에서공동연구를진행하는경우에동일한샘플을사용하여다양한유전자발현검출시스템이나분석방법을사용해야하는경우가있는데이경우에는샘플의균질성 homogenecity 을유지해야한다. Ovation 으로증폭한 c는일관성consistency이있으며, 안정적이고표준화되어있어샘플양이적고한정되어있는샘플을공동연구에사용하고자할때적합하다. 이보고서에서는 qpcr을이용하여 Ovation RNA Amplification V2 (Code 31) 의효율을나타내었고, FL-Ovation c Biotin Module V2 (Code 42) 를사용하여 GeneChip array 분석결과를보여주고있다. 이연구에사용한 RNA는다음과같다. HeLa cell line RNA (Ambion, Cat. # 7852) UHR total RNA (Stratagene, Cat. # 74) Spleen total RNA (Ambion, Cat. # 797) Placental total RNA (Ambion, Cat. # 795) Ovation RNA Amplification V2 (Code 31) 를사용하여모든 RNA를증폭하였으며, 실험프로토콜은제품사용자가이드에서설명한절차에따라진행하였다. Real Time PCR 실험은 Universal ProbeLibrary와프라이머제작소프트웨어를이용하여설계하였다. 프라이머는 Integrated Technologies (Coralville, IA) 에의뢰하여합성하였으며, multiple assay의경우프라이머를각유전자의 5 부분, 중간부분, 3 끝부분에디자인하여진행하였다. 데이터선택기준은 efficiency 가 1% 에가깝고, slope가 인경우로하였다. 실험에사용한프라이머및 probe의정보와 accession number는요청시제공가능하다. qpcr 반응에는단순히증폭한 c를정제없이 1/1로희석 (1 X TE (PH 8. ) 이용 ) 한 c와정제한 c 2 ng을사용하였으며, 사용한프라이머와 probe는아래와같다 nm forward & reverse primer, 1 nm ProbeLibrary probe 2. Assays-on-Dem primer probe mix (ABI) 반응시약은 TaqMan Fast Universal PCR master mix를사용하였으며, 최종반응양은 2 μl로하였다. qpcr 기기는 ABI75의 fast block installation 을사용하였으며반응은기본프로토콜로진행하였다. GeneChip array 분석을위하여, 증폭한 c의단편화과라벨링에는 FL-Ovation c Biotin Module V2(25μl반응에 3.75μg의 c이용 ) 를사용하였으며, array는 HG-U133A 2.. GeneChip arrays (Affymetrix, Cat.# 9469) 를이용하였다. 실험에사용한 c 최종농도는 17 ng/ μl였다. Array 결과는 Affymetrix GCOS stware(genechip Operating, ) 를이용하여분석하였다. 3 Life Science & Biotechnology 48

33 NGS Microarray Applications Total RNA 정량은 Nanodrop ND-1 spectrophotometer (Wilmington, DE) 을사용하였으며, 증폭한 c 검정은 RNA 6 Nano LabChip (Agilent Cat.. # ) 과 Eukaryotic Total RNA Nano program (Nano assay in Expert 21 stware) 을이용하였다. 결과기 RNA양에상관없는재현성과최고의증폭효율 5, 2, 5 ng의 total HeLa RNA를사용하여증폭한 c를 Agilent Bioanalyzer를이용하여비교하였고, 그결과기 RNA양에상관없이유사한양상을나타내었다 ( 그림 1). 이 c를정제한 c와정제하지않은 c로나누어 7개의유전자의발현을 qpcr로분석하였다. 이결과또한기 RNA양은물론, 전사산물transcript의양에상관없이매우민감하고재현성이높게증폭되었다 ( 그림 2). 증폭된 UHR과 HeLa RNA의 c 양은그림 2, 3에표기하였다. 다른조직에서추출한 RNA를이용하여 c를증폭한경우에도일정하게 4~8 μg정도의 c가증폭되었고, RNA를추출하기어려운태반placenta이나전혈whole blood에서도최소 4~5 μg의 c가증폭되었다. 제품 lot에상관없이재현성과최고의증폭효율 Ovation RNA Amplification V2의 lot에따른결과를비교하기위해서로다른 3개 lot을사용하여실험을진행하였다. UHR RNA(5 ng) 를 Ovation RNA Amplification V2를사용하여증폭하고, 2반복duplicate으로 5개의유전자의발현량을비교하였다. c 증폭량과 3개 lot의 qpcr 결과는거의일치하였으며, 재현성도나타내었다 ( 그림3). Ct Reproducibility qpcr yields across lots samples GAP33 GAP1. TFRC.5 TFRC1. b-act M YIELDS (µg) Gene 그림 3. qpcr yields reproducibility across multiple product lots. 5 ng UHR total RNA was amplified in triplicate for 3different lots Ovation RNA Amplification V2. qpcr results for 5 genes show reproducible consistent amplification. Amplified c yields, shown on left axis graph, also show very consistent amplification across multiple lots. Fluorescence Migration 5 ng HeLa 2 ng HeLa 5 ng HeLa 25b 2b 5b 1kb 2kb 4kb 6kb RNA 6 ladder 그림 1. Size distribution amplified c across total RNA input. Amplified c using 5, 2, 5 ng total HeLa RNA show consistent prile size distribution across RNA input range upon analysis Agilent Bioanalyzer traces. 증폭된 c는일관된 fold change를보임태반, 비장spleen 조직을이용하여다중혼합 RNA를준비하였다 ( 자세한내용은별도문의 ). c 증폭에는 1% 태반 RNA 5 ng과태반 / 비장 RNA(5 : 5) 5ng이사용되었다. c 증폭후에 qpcr을이용하여태반에서만발현되는 18개의유전자를분석하여 fold change를비교하였다. 2-fold change는 1% 태반 RNA와 1대1로태반과비장 RNA가섞인 RNA를비교하였을때나타날것으로예상되었다. qpcr 실험결과대부분의유전자에서약 2배차이를나타냈다 ( 그림 4). 에러바의높이가일정한것은증폭한샘플에서일정하고정확하게 fold change가일어났음을보여주는것이다. RPL35-1은 Ct 값의 nomalizer로사용되었으며, M4-2, DAD1-1, SEPT2-1은 housekeeping 유전자로사용하였고, 이유전자들은비장에서조금더높은발현을보였다. Ct Value Reproducibility qpcr yields across RNA input purifiction (Amplified c; unpurified purified) 5 ng, unpurified 5 ng, purified 2 ng, unpurified 2 ng, purified 5 ng, unpurified 5 ng, purified Fold Change Fold Change is cosnsitent across many genes expect a 2 fold change (1% Placenta vs. 5% Placenta) KRT 19-1 HSD 3B1-3 CGA-1 ALPP-1 C YP2J2 EPS8L1-4 IG F BP1-3 FN1-2 PSG 11-2 LRP2-2 ISL1-1 GCM 1-2 Gene F LJ PP TAC3-2 GPC 3-2 ADAM12-F3 PAG E4-1 M4-2 DAD 1-1 SEPT2-1 R PL NONO-3 RPL35-1 DAD-1 EIF4G2-5 EIF4G2-3 TBP-2 MYST-3 YIELDS (µg) Gene 그림 2. qpcr yields reproducibility across total RNA input transcript abundance. 5, 2, 5 ng HeLa total RNA were amplified in triplicate. qpcr results for 7 transcripts show reproducible consistent results with without purification amplified c. Yields, shown on left axis graph, also show highly reproducible degrees amplification across inputs 그림 4. qpcr data shows expected consistent fold change: Using a tissue mixing model, amplification 5 ng a 1% Placenta RNA sample vs. a 5% mix Placental Spleen RNA consequent qpcr assays a panel 18 Placenta-specific genes showed that expected 2 fold changes observed maintained across different transcripts. The rightmost 4 genes in graph are housekeeping genes, very rightmost gene, RPL35-1was used as a normalization gene. 31

34 NGS Microarray Applications 높은 array metrics 와높은 signal correlation, call concordance 를나타냄. 5 ng의 HeLa RNA를 2회증폭하여얻은 c를다편화와라벨링과정을거쳐 GeneChip array에 hybridization시켰다. 그림 5의표에 array의결과를나타냈다. Scaling factor, Background, % present calls, 3 /5 비율등이모두허용가능한수준으로나타났다. 그림 6에서는 5 ng HeLa RNA를이용하여 GeneChip array 결과를분석한결과, 중복duplicate 실험에서.994의 signal correlation과 93.2% 의 call concordance 값을보여주었다. 이결과는증폭된 c 타겟이높은수준의재현성과일관성을가지고있음을보여준다. Raw Q.97 Scaling Factor.69 Back- ground 34.5 % Present 64.1 (3'/5') GAPDH 1.3 3'/5' Actin 그림 5. Array metrics for duplicate samples : 5 ng HeLa RNA, amplified, fragmented Figure labeled 5. in Array duplicate, metrics hybridized for duplicate to HG-U133A samples: 2. GeneChip 5 ng arrays, HeLa show robust reproducible array metrics. R 2 =.994 Call Concordance= 93.2% 그림 6. High reproducibility demonstrated by signal correlation call concordance : 5 ng HeLa RNA, amplified, fragmented labeled in duplicate, as previously described show high signal correlations R call concordance 93.2% demonstrating very high level reproducibility between independently processed samples. 결론 Ovation RNA Amplification V2를사용하면 5 ng의매우적은 total RNA를이용해서도재현성높은증폭을할수있다는것을보여주었다. 이와같이적은양의 RNA만필요하기때문에극미량의샘플, 희소가치가높은샘플, 샘플중에극소량만사용해야하는실험등에쉽게적용할수있다. 특히대체할수없는제한적인임상샘플분석에매우중요한툴로사용할수있다. 간단하고, 빠르며, 자동화과정에쉽게적용할수있는 Ovation의특징은높은감도와특이성높은 c를증폭시켜우수한 array 결과를얻을수있어대형유전자발굴실험과임상연구에이상적이다. 32 Life Science & Biotechnology 48

35 NGS Microarray Applications Technical Note 2 WT-Ovation FFPE WT-Ovation FFPE 을이용한폐조직 FFPE 샘플과신선동결샘플의발현프로파일링비교 서론 FFPE(formalin-fixed paraffin embedded) 조직으로부터추출한 RNA를이용하여발현분석하는것은힘들고어려운일이었다. 그이유는시료처리과정과조직간의 cross-linking을일으키는고정과정 fixation, 이조직들로부터추출한핵산의분해와같은지극히극단적인조건들때문이었다. WT-Ovation FFPE 은심각하게분해된한정된 RNA를증폭함으로써이와같은조건들을극복하였다. 이시스템으로인해소중한샘플의전체적인유전자발현분석을위한기술적인한계를극복할수있게되었으며, 보관하고있는대량의 FFPE 샘플과그와관련된임상데이터를활용할수있게되었다. FFPE에서추출한 RNA를이용해유전자발현의가치를평가할때가장중요한고려사항은이결과가생물학적으로중요한정보인가를판단하는것이다. 이질문에대한답을위해서, 본내용에서는 FFPE에서추출한 RNA와동일한조직의신선동결샘플fresh frozen sample로부터추출한 RNA의발현양상을비교하기위하여, 폐암조직human lung tumor 과정상인접조직normal adjacent tissue을 FFPE와신선동결샘플fresh frozen sample로만들어, 각각에서추출한 RNA를분석하였다. 재료및방법 RNA 샘플암조직과정상인접조직의신선동결샘플과 FFPE 샘플, 각각으로부터 Formapure Kit (Agencourt, Cat. #A33341) 또는 Qiagen RNeasy mini kit (Qiagen, Cat. # 7416) 를사용하여제조사의매뉴얼에따라 RNA를추출하였다. c 증폭과 Array Hybridization 모든증폭은 WT-Ovation FFPE (Code 34) 을이용하여제조사의방법에따라진행하였다. 신선동결샘플에서추출한 RNA는 1 ng, FFPE에서추출한 RNA는 5 ng을기샘플로사용하였다. GeneChip array를이용한유전자발현분석을위한샘플준비는 FL- Ovation c Biotin Module V2(Code 42) 를이용하였으며, 증폭한 c 5 μg를사용하였다. 이제품을이용하여 fragmentation과 label 한 c를 HGU133A 2. Affymetrix GeneChip arrays에적용시켰으며, streptavidin phycoerythrin 과항체증폭antibody amplification 으로염색한후제조사의프로토콜에따라스캔하였다. Array 결과는 Expression Console (Affymetrix), BioConductor 1.9 package와 Partek Genomics Suite를이용하여분석하였다. 경로pathway 와기능적인분석은 Ingenuity Pathway Analysis stware를이용하였다. 데이터분석동결조직샘플은 3회반복, FFPE 조직샘플은 4회반복실험을하였다. GeneChip array에서 RMA intensity value와 MAS5 PMA call을얻었고, pair wise tumor-normal Student t-test와관련된 p-value 는 Bioconductor 1.9를이용하여결정하였다. 최소한 4개 Present call 중 3개, 3개 Present call 중 2개의 probe 세트를 present call overlap analysis에포함시켰다. 유전자들은 FFPE 와 Frozen tissue 모두에서암조직과정상조직에서서로다른발현을나타냈다. 발현의차이를나타내는유전자의기준은 RMA value가 2배혹은그이상차이가나고, pair wise t-test의 p-value가.1 이하인것으로하였다. 서로다르게발현된유전자는 Ingenuity Pathway Analysis를이용하여분석하였다 ( 그림 2). 결과 FFPE 조직에서추출한샘플을이용한 array 결과는표 1A에나타냈다. 각샘플의 present 라고나타난 probe 세트의퍼센트를이용하여감도를평가하였다. FFPE 샘플의 present call 수가신선동결샘플의수보다적음에도불구하고, 증폭분석은매우용이하여, 매우광범위하게많은수의유전자를검출하면서약 5% present mark에가깝게분석할수있었다. Array 재현성은표 1B에정리하였다. A B Sample Type N Avg. SF (sd) Avg. Bkgd (sd) Avg. %P (sd) Frozen Normal (.6) 3.1 (.6) 66.2 (1.1) Frozen Tumor (1) 3.7 (1) 62.6 (2.8) FFPE Normal (.6) 29.6 (.7) 49.9 (1.3) FFPE Tumor 4 13 (.6) 29.5 (.5) 48. (1) Sample Type Frozen N Frozen T FFPE N FFPE T Frozen N.99 Frozen T FFPE N FFPE T Table 1. A. Affymetrix HG-U133A 2. array performance metrics. Listed are number arrays (N), 표 1. A. Affymetrix HG-U133A 2. array performance metrics. Listed are number arrays (N), Scaling Factor (SF), Background (Bkgd), Percent Present calls (%P) with stard deviation listed in parensis (sd) for each value. B shows array average Pearson correlation matrix, demonstrating high level reproducibility between technical replicates. 33

36 NGS Microarray Applications FFPE 샘플의 present call probe 세트는신선동결샘플에서도동일하게검출되었지만, 신선동결샘플에서보다많은 present call probe 세트가검출되었다. FFPE 샘플에서 RNA 분해의가능성과신선동결샘플에서보다많은 present call이나타날것이라는것은예상되었던결과이다 ( 그림 1). FFPE 샘플에서몇개의유전자발현이특이하게관찰되었지만전반적인발현패턴에는영향을주지않았다. # arrays %P Background signal Scaling factor c Yield µg QC metric 기능적인발현부분이일치함을확인하기위해, 서로다르게발현된유전자세트를 Ingenuity Pathway Analysis를이용하여분석하였다. 각각의 pool은개별적으로분석되었다 ( FFPE T > N, FFPE N > T, 신선동결샘플 N > T, 신선동결샘플 T > N ). Canonical pathway analysis 는서로다르게발현되는유전자에서통계학적으로대표되는유전자를이용하여신선동결샘플에서 11개의 pathway 를밝혀냈다. FFPE 샘플에서는 11개 pathway 중 9개 pathway 를통계학적으로유사한수준으로밝혀냈다. 2개 pathway 는 FFPE 샘플에서낮은수준으로유일하게검출되었으나, 통계학적으로는신뢰도가있었으며, 그결과는그림 3 에나타냈다. 일반적으로유전자를그룹화할때많이이용되는생물학적방법으로분석했을때, 암조직에비해일반조직에서 upregulated되는상위 16 개 cellular process ( 세포내과정 ) 중 15개가신선동결샘플과 FFPE 샘플모두에서관찰되었다. 단하나의유전자그룹이 FFPE 샘플에서특이하게검출되었으며, 그림 3B에표시하였다 ± ± ± ±.88 그림 1. Venn diagram concordance present calls between FFPE Frozensample for both tumor (T) normal (N) sample sets. Total number present calls is on bottom, middle number in circle overlap corresponds to concordant calls, present calls unique to each subgroup are in circles above its respective name. 그림 2에서는 Venn diagram으로암조직과정상조직사이에다르게발현되는유전자를정리하였다. 신선동결샘플에서다르게발현된 5 % 이상의유전자들은 FFPE 샘플에서도동일하게검출되었다. 결과선별기준은 p-value는.1 이하, differential expression value는 2배이상으로하였다. 이렇게서로다르게발현된유전자들을확인하기위해서정상조직과암조직에서 4회반복으로실험하였으며, 이레벨은거의모두일치하였다 A 14 Significance Scores B 3 Significance Scores FFPE versus VS Fresh Fresh Frozen Frozen Pathway Pathway Analysis Analysis Fresh Frozen Functions FFPE Functions Complement & Coagulation Cascades Antigen Presentation Nicotine Nicotinamide Metabolism NF-kb Signaling IL-1 Signaling IL-6 Signaling Leukocyte Extravasation Signaling Eicosanoid Signaling Death Receptor Signaling PPAR signaling Toll-Like Receptor Signaling Histidine Metabolism PDGF Signaling Pathways FFPE VS versus Fresh Fresh Frozen Frozen Functional Analysis Analysis Fresh Frozen Functions FFPE Functions Im m une R espon se C ell-c ell Signaling Interactions C ellular M o vem ent H e m atological S yste m D evelopm ent & M a in t. Inflam m ato ry D isease Im m u ne Lym p hatic S ystem D evelopm ent Tissue D eve lo pm ent C onnective Tissue D isorcers Skeletal M u scu lar D isorders O rgan ism al Injury A bnorm alities Tissu e M orp hology C ellular Growth P roliferation Cell Death C ancer Functional Categories # 1 Figure 1. c amplification yields 41 FFPE-derived Ovarian tumor samples are shown. on GeneChip arrays, a criteria me by all amplification 그림 3. Ingenuity pathway analysis differentially regulated canonical pathways (A) cellular functions (B) in tumor normal tissues in Frozen FFPE datasets. Pathways cellular functions are listed along X-axis. The Y-axis represents log(significance score), which is defined by Ingenuity depends on number genes (components) involved in pathway/process ir relative weight in process. The orange line represents significance threshold. Nine eleven canonical pathways represented in Fresh Frozen samples were detected in FFPE samples. 그림 2. Venn diagram concordance differentially expressed genes between FFPE Frozen samples. Genes are called differentially expressed on Affymetrix HGU133A 2. between tumor normal in FFPE Frozen samples if y meet following criteria: 2x or greater RMA expression value difference p-values lower than.1 from a pair wise t-test. There were only 4 false positives in FFPE comparisons (1 int>n, 3 in N>T). 34 Life Science & Biotechnology 48

37 NGS Microarray Applications 결론 WT-Ovation FFPE 을 FL-Ovation c Biotin Module V2 와같이사용하면 FFPE 조직에서추출한심각하게깨진 RNA에서도전반적인유전자발현프로파일링과 microarray 분석이가능하다. 본고에서는높은품질의신선동결 RNA를사용한것과비교하여 FFPE 샘플로부터만들어진유전자발현정보의가치를확인하였다. FFPE RNA를이용했을때민감도가감소가할것을예상했음에도불구하고, FFPE 샘플과신선동결샘플을이용한 differential expression 결과는매우일치했다. 매우높은유사성은아래와같다. FFPE 조직시료와같이극한조건의 RNA로부터도발현프로파일링이가능하도록기술이개발된다면, 가지고있는 FFPE 조직시료로부터얻은유전자발현프로파일을임상결과와연결시킬수있고, 이는매우가치있는것이될것이다. WT-Ovation FFPE 제품은임상관련유전자의발현특징을발굴하고확인을가능하게하는진보된기술중하나이다. Array상에서관찰된전사산물 transcripts 서로다르게조절된다고밝혀진전사산물 transcripts FFPE와 fresh frozen 샘플결과를비교했을때밝혀진생명활동 (pathway & functions) NuGEN s Portfolio NGS Products..to enable simple, fast, scalable NGS workflows 1 35

38 NGS Microarray Applications Real Time PCR 이전단계인 RNA 증폭에대한 FAQ 1. qpcr도증폭인데, 왜전단계에서증폭해야하나요? 생검biopsies, LCM(Laser Capture Microdissection), cell sorting과같이작은샘플에서추출한 RNA 양은매우한정적입니다. 좋은 qpcr 실험디자인은다양한종류의컨트롤과 3회반복실험을필요로하기때문에충분한양의 RNA가필요합니다. 그러므로관심있는유전자의수가증가함에따라샘플양도증가합니다. 게다가발현량이적은유전자들은일반적으로검출레벨이낮고, 이러한한계는양이적고구하기힘든샘플에서는보다더중요하게대두됩니다. qpcr 분석전에 RNA를증폭하면발현량이적은샘플들을검출가능한범위로올려, 높은민감도와재현성높은결과를얻을수있습니다. 그러므로 RNA 증폭은적은생물학적인샘플에서충분한양으로많은유전자에대하여보다좋은디자인, 유익한정보를주는 qpcr 검출을가능하게합니다. 3. 관심있는유전자가적은데, 왜 RNA를증폭해야하나요? 수행하고있는연구가한정된수의유전자에집중되어있다면현재는증폭의필요성을느끼지못할지도모릅니다. 그러나과학은매우빠르게새로운기술과발견, 조사와분석적인접근방법이발전되고있습니다. 예를들어, 과거에분석하지못했던새로운 array 데이터를당신이관심갖고있는분야나새로운논문에서발견하게될수도있습니다. 당신이 c 샘플을증폭한다면, 원래 RNA 샘플을보다더안정적이고풍부하게합성할수있습니다. 이 c를이용하면증폭한샘플과데이터를다시만들필요가없으며 RNA 샘플의추가적인소모없이주어진시간내에연구할수있습니다. 따라서 c합성은유전자의수를늘리면서미래의 qpcr 응용연구에적용하기위해보존할수있는유용한단계입니다. 2. 샘플양이많은데, 증폭해야할이유가있나요? 물론있습니다. 예를들어전체기관Whole organ과같이샘플양이많다는것이 qpcr에사용할수있는샘플이많다는것을의미하는것은아닙니다. 첫번째, 유전자발현연구의주요목적중하나는관찰된표현형이나병리학적현상과의발현패턴의연관성을알아보는것입니다. 이목적은가능한한동일한샘플을이용하는것이가장좋은결과를얻을수있습니다. 전체기관이나다른기관의많은부분에있는서로다른분포의세포는발현프로파일링이겹치거나낮은분석능resolution을나타낼수있으며, 다양한소스로부터유래한 RNA를이용하여실험할경우표현형과발현프로파일링관계를민감하고, 명확하게연결시키기힘들수도있습니다. LCM, cell sorting, 그외다른세포집단분류 cell population classification 등에대한접근기술이발전하면서, 대부분의샘플은동일한세포의모집단을모을수있어서보다좋은하위분류sub- classification가가능합니다. 이것은 qpcr을이용한유전자분석에서보다소중하고정확한생물학적인결론을가능하게할수있습니다. 두번째, mrna 레벨에서유전자의 differential expression은반응상에서다음에있는경로pathway의첫번째지표인경우가있습니다. 이러한지표뒤에정확한생물학적인연구를위해서단백질발현, 단백질변형modification, in situ hybridization 혹은면역염색법중의하나로꼭검증해야합니다. RNA 증폭은원래샘플을활용하여 downstream 연구를진행할수있게해줍니다. 게다가보다큰생물학적인샘플은종종다양한세포타입과하위세포타입이혼합되어있는경우가있습니다. RNA 증폭은같은조직샘플에서다른세포타입에대한연구를분리해서가능하게할수있으므로, 절개dissection나 cell sorting에의한세포타입분리도가능하게합니다. 4. 실험디자인에 RNA 증폭이주는가장큰장점은무엇인가요? RNA 증폭은실험디자인에중요한영향을줍니다. 현재당신은 qpcr을위한많은샘플을가지고있으므로생물학적샘플에대한요구는덜할것입니다. 하지만다음스텝의연구디자인을위해 RNA 증폭의유무를고려하세요. 증폭을하지않으면, 연구를위한적당한양의샘플을확보하기위해샘플에대한 pooling이필요하거나많은수의샘플을수집하고만들어야합니다. 증폭을하면 -. Pooling에의한나타날수있는내재되어있는생물학적인다양성을줄이면서양이적은샘플에대한연구를할수있습니다. -. 예를들면실험동물과같이수집하고만들어내야하는샘플의수를줄일수있습니다. -. 수집한샘플내의하위모집단subpopulation과특정한세포타입에대한연구를할수있습니다. 그러므로연구의민감도를증가시킬수있고보다중요한생물학적인데이터를얻을수있습니다. 증폭을하지않으면, 구하기어렵고양이적은임상샘플은제한적인연구에만사용될수밖에없습니다. 36 Life Science & Biotechnology 48

39 NGS Microarray Applications 증폭을하면 -. 한정된샘플로부터충분한양의 c를합성할수있으므로, 당신의연구는물론공동연구자들과나누어사용할수있는충분한샘플을확보할수있습니다. 그러므로원래의 RNA 샘플을미래의연구를위해남겨놓을수도있습니다. -. 극도로한정되어있고구하기힘든환자샘플을충분히활용할수있습니다. 예를들어당신이갖고있는단 5 ng의샘플은 5 μg의가치를가질수있습니다. -. 구하기어려운샘플의데이터와재료를공동연구자들과나눠사용할수도있는샘플뱅크컨소시엄을구성할수도있습니다. -. 관심있는유전자각각에대하여같은발현범위에서충분한내부컨트롤internal control 실험을할수있습니다. -. 실험시스템에대한적당한 normalizing 유전자를결정할수있고, 선택에따라많은 normalizing 유전자를사용할수있다. -. 다양성이줄어든상용컨트롤 RNA가아닌직접실험샘플을이용하여 qpcr 조건을최적화할수있습니다. 증폭을하지않으면, 한정된샘플양을기본으로하여디자인해야합니다. 한정된샘플양은실험디자인의질적인면에서중요합니다. 증폭을하면 -. 당신의모든연구에서생물학적기술적모든부분에서필요한만큼반복실험을할수있습니다. 실험가설 뇌종양연구를위하여마우스모델시스템의 3개의단계에서 tumor development 와 RNA 발현을연구하고자한다. 최단계의샘플로부터는약 5 ng의 total RNA를추출할수있고, 그다음단계에서부터는약 1 ng의 total RNA를추출할수있다. 실험은 3회반복으로진행하고 qpcr은 2개의 housekeeping gene과 2개의타겟유전자를증폭한다. 반응당 total RNA 1 ng을사용하는단일반응으로진행한다. 증폭방법을이용하지않는가설모델 1.. 이연구에서는총 22개유전자의발현을조사한다. 이를위해최단계에서는 마리의마우스가필요하고, 그후단계에서는마우스 7-8 마리가필요하다. 따라서총 28-3 마리의마우스가필요하다. 2..qPCR을수행한후결과를분석한다. 3..이연구에서매우유의성있는결과를얻었다. 몇몇단백질과그단백질의위치localization가관여될것이라는가설을세운다. 4.. 새로운마우스를이용하여단백질위치분석을해야한다. 단백질위치분석을한다. 5.. 단백질위치분석이이가설을증명한다. 그러나여기에아직도추가로다른유전자들이관여되는지의문이생긴다. 따라서새로운후보유전자를밝혀내기위하여 microarray 실험을하기로결정한다. Microarray 실험은각단계에서 3회반복실험을한다. 따라서총 9 마리의마우스가필요하다. qpcr 분석에이은단백질위치분석을통해이와같은현상을이해했으므로, 이와동일하게또다른 24 마리의마우스가필요하다. 조직샘플은추출해서 -8 에보관한다. RNA 데이터와단백질데이타를직접적으로비교할수없다. 6.. 새로운유전자에대해단백질위치와면역침강반응은물론 qpcr 실험을진행한다. A 증폭방법을이용하는가설모델 1.. 이연구에서는 22개유전자의발현을조사한다. 각단계에마우스 3 마리씩이필요하다. 총 9 마리의마우스가이실험을위해사용된다. 2.. qpcr을수행한후결과를분석한다. 3.. 이연구에서매우유의성있는결과를얻었다. 몇몇단백질과그단백질의위치localization가관여될것이라는가설을세운다. 4.. 새로운마우스를이용하여단백질위치분석을해야한다. 단백질위치분석을한다. 5.. 단백질위치가이가설을증명한다. 그러나여기에아직도추가로다른유전자들이관여되는지의문이생긴다. 따라서새로운후보유전자를밝혀내기위하여 microarray실험을하기로결정한다. Microarray 실험은각단계에서 3회반복실험을한다. 따라서총 9 마리의마우스가필요하다. 단백질위치분석을통해이와같은현상을이해했으므로, 조직샘플은추출해서 -8 에보관한다. 6.. Microarray 샘플의남아있는 c를이용하여새로운유전자에대한 qpcr 분석을실시한다. 조직의남아있는부분을이용하여단백질위치분석과면역침강실험을하고, 이결과를 qpcr 결과와직접적으로연결시켜분석한다. 만약결과가연관성이있다면, qpcr에는첫번째세트의마우스를사용할수있다. 추가로 RNA 를추출할필요가없으므로, 시간을줄일수있다. 이연구에서 qpcr 전에 c를증폭하는장점 1.. 비용을줄일수있다. : 실험동물이 3-4배정도적게필요하다. 2.. 시간절약 : 같이증폭한 c를 microarray와 qpcr 실험에사용할수있다. 3.. 단백질과 RNA 결과를직접적으로연결할수있다. 4.. 반복실험의총횟수를증가시키면서동물의개체간의 RNA 데이터를비교할수있다. 5.. 충분한실험재료를미래의 qpcr과 array 실험에사용할수있도록남겨둘수있다. 37

40 SMARTer Ultra Low RNA Kit for Illumina Sequencing Clontech 과 Illumina 의기술결합으로극미량의 RNA 염기서열분석가능 Illumina sequencing 프로토콜에추천제품 뛰어난민감도sensitivity - 1 pg의 total RNA 적용가능 1개의 tube에서반응수행 - 샘플보존 RNA-seq 에적용 - whole transcriptome 정보분석에유용 Single-cell analysis - 세포로부터곧바로 RNA-seq library 제작가능 RNA Sequencing(RNA-Seq) 은다양한세포의 transcriptome complexity를분석할수있을정도로유전자발현분석의혁신을이루고있다. 그러나기존의 RNA-Seq 방법이유전자발현분석을위해요구되는기 RNA 양이적어지기는했으나분석하고자하는시료가매우제한된극소량일경우여전히적용이어렵다. 줄기세포 stem cells, 순환하는암세포circulating tumor cells과뇌조직brain tissue biopsies 와같이희귀하고중요한시료를연구하기위해서는극도의민감성과재현성이보장되는방법이필요하다. 이한계를극복하기위해 Clontech의 SMART 기술과 next-generation sequencing 기술의선두주자인 Illumina가협력하여극도로제한된시료를분석하기위한강력한방법을제공하게되었다. SMARTer Ultra Low RNA Kit for Illumina Sequencing(Code ) 은 1 pg의극미량의 total RNA 로부터간편하고효율적으로 library를제작한후 Illumina사의 HiSeq, HiScanSQ 과 Genome Analyzer IIx 로분석하도록가이드하고있다 (Illumia사권장프로토콜에포함 ). 본 Kit로합성된 c 는변형된 Illumina library prep 프로토콜을이용하여표준 Illumina sequence-by-synsis (SBS) sequencing에이용할수있다. Total RNA c synsis w/t SMARTerUltra Low RNA Kit Modified Illumina Library Prep Protocol Cluster Generation Sequencing on Illumina HiSep. HiSon. or GAllx Analysis SMARTer Ultra Low RNA Kit Illumina Sequencing Workflow. QC c How Much RNA is in a Single Cell? nerve cells blood cells epilial cells 세포로부터추출되는 RNA의양은세포의크기나대사상태에따라큰차이를보인다. 그러나일반적으로신체세포대부분은 1-3 pg의 RNA를포함하고있다. SMARTer Ultra Low RNA Kit for Illumina Sequencing은소수의세포로부터추출된미량의 RNA 로부터도고품질의 RNA sequencing 결과를얻을수있도록하는강력한기술이다.

41 Methylation Studies. Bisulfite EpiXplore Methyl Detection Kit RE Digestion Methylation Sensitive Restriction Enzymes Methylated Enrichment EpiXplore Methylated Enrichment Kit For Individual CpG Loci For Genome-Wide! MSP (qmsp) EpiScope MSP kit PCR TaKaRa Taq Hot Start Version Real Time PCR SYBR Advantage GC EpiScope Promoter qpcrarray (Human) Amplification RE Digestion Cloning HTP Sequence Microarray Restriction enzymes Regents Kits for cloning ChIP-seq, etc CpG Isl Microarray Promoter Microarray Electrophoresis (COBRA) Regents for gel electrophre sis Sequence (Bisulfite Sequence) Control EpiScope Methylated HeLag EpiXplore Methylation Detection Kit EpiXplore Methylated Enrichment Kit Exceptional sensitivity with only 5 pg starting material required equivalent to only 8 mammalian cells. Cytosines (C) are converted to Uracil (U). 5-methyl-cytosines (C) are unreactive remain as cytosines after bisulfite modification. Couple MBD2 protein to beads (1 hr). Input genomic! TALON! TALON! MBD2! Sheared Genomic! Me! Sulphonation! Deamination! Desulphonation! HSO -! H 2O! NH +! 4 C! 3 OH -! U! OH -! HSO -! 3 Capture methylated TALON! on MBD2-Beads (1 hr). Unbound! Enriches for over 95% methylated CGCGTCTATG CGAGGCCGG CGCGTCTATG CGAGGCCGG Bisulfite modification Elute methylated TALON! Enriched is immediately ready for downst ream applications CGCGTUTATG CGAGGUCGG UGUGTUTATG UGAGGUUGG PCR Amplification CGCGTTTATG CGAGGTCGG GCGCAAATAT GCTCCAGCC TGTGTTTATG TGAGGTT GG ACACAAATAT ACTCCAACC Mrthylated! Protocol allows for gradient separation low, medium highly methylated EpiScope MSP Kit Most widely used method for methylation Easy to use with no restriction digests or Sourn blots required. Methylated HeLa genome! Native HeLa genome! Easy to analyze multiple samples comparison. EpiScope Flexibility experiment with a universal method for any gene interest. MSP Sensitive detection allows use as little as 1 ng A Optimized kit for MSP High amplification efficiency with bisulfite treated (up to 2 bp) High specificity for identification methylated/unmethylated CpG Sufficient quantitative analysis to detect 5% difference methylation on alleles Compatible with both real time PCR end point PCR B C D M specific primer UM specific primer

42 Mammalian Inducible Expression Endless Clontech Possibilities is exclusive provider idimerize products inducible dimerization systems cell- permeant ligs which were previouslyprotein provided by ARIAD Pharmaceuticals, Inc. under br idimerize Inducible Dimerization name ARGENT. This technology has been used by over 2, investigators in 35 countries, with over 4 scientific publications to date. Clontech이 새롭게 공급하고 있는 idimerize 제품(구 ARIAD사의 Dimerizer(dimerization의 화학적 유도물질)는 세포 투과성의 유기 Why Manipulate Oligomerization State Proteins? 의 연구자들이 사용하고 있다. 저분자 물질로 두 개의 분리된 모티프motif를 갖고 있다. 이 모티프는 왜 단백질의 Oligomerization을 조절하는가? Many critical processes in cell require protein oligomerization. In fact, majority human proteins can form 단백질의 oligomerization은 많은 생리 변화 과정에 필요 oligomers including most세포내 cell surface receptors >7% 하며, human 실제enzymes. 인체 단백질의 대부분이 oligomer를 형성하며 세포 표 provider idimerize products inducible dimerization systems cell- 면의 receptor나 7% 이상의 효소가 oligomer를 형성한다. dimerization technology canbr be applied to any ere previously provided byinducible ARIAD Pharmaceuticals, Inc. under Inducible process dimerization 기술을 단백질의 infl 상호작용이나 biological that can be 활용하면 manipulated ology has been used by over 2, investigators in 35 countries, with overby 4 uencing interactions/localization a protein.과정을 연구하는데 활 발현 위치localization를 조작하여생물학적인 ate. te idimerize는 어떻게 작용하는가? ARGENT 브랜드)은 이미 4편 이상의 논문과 35개국 2명 이상 용할 수 있다. Inducible dimerization means small molecule control pathway activity, or location your 활성 protein Inducibleinvolvement, dimerization은 목적 단백질의 pathway나 또는 interest. Oligomerization Proteins? 위치 등에 대한 State 조절을 저분자를 이용한 제어하는 것을 의미한다. cell require protein oligomerhuman proteins can form ll surface receptors >7% ogy can be applied to any manipulated by influencing a protein. (1,e2)transcription C과 ell같은 signa세포내 ling 활성을 모방하게 된다 Gen. 반대로 reverse dimerizer A poptosis뭉쳐있던 단백질에 결합하여 Enz단백질끼리 yme activat분리되도록 ion lig는 한다. Protein secretion Protein relocalization Pathway 저분자에activation 의한 세포신호전달 Cell adhesion Protein synsis 조절 Cell rolling 많은 신호전달 Protein splicing체계는 대부분 신호전달 RNA 단백질의 splicing 상호작용에 의해 활 성화 외부 자극에 반응한 표면 G lyco된다. sylatio세포 n DN세포 A loo ping수용체 단백질은 세포 내의 신호전달 이것으로 전사가 유도되거나, Neurite growth 단백질을 활성화 한다. Transformation Gene transcription 이펙터 단백질 분비된다. 이 신호전달의 Amyloid fibril formation Substitute your research effector protein이 활성화되거나 interest here lig가 인식하는 어떠한 과정에서든지 dimerizer를 통해 dimerizer Enzyme activation domain을 갖고 있는 융합 단백질에 의해 조절 가능하다. Table 표 1 I: Just Some Published Processes Controllable by idimerize Technology Cell signaling Apoptosis How Does idimerize Work? Protein secretion small molecule control or location your protein A 목적 유전자에 결합된 특정 단백질 모듈(Dmr domain)에 대해 높은 친 Table I:있어 Just결합하게 Some Dimerizer를 Published Processes Controllable 화성이 된다. 첨가하면 chimeric protein by idimerize Technology subunits이 서로 매우 근접하게 되어 목적 단백질의 dimerization Protein relocalization Pathway activation Protein synsis Cell adhesion Cell rolling idimerize Inducible Homodimer (Code 63568)은 동일한 binding 갖고 Homodimerizer lig를 이용 Protein splicing RNA splicing chemical inducer dimerization, or dimerizer, is a cell-permeant organic smallmotif를 molecule with있는 twob/b separate motifs that each bind with Glycosylation D N A l o o p i n g 하여 single signaling domain이나 다른 관심있는 단백질의 selfc protein module (Dmr domain) fused onto protein(s) interest. Addition dimerizer brings chimeric Neurite growth Transformation protein subunits into very close proximity to each or, mimicking activation 유도한다. cellular event that dimerization protein association을 Amyloid fibril formation Substitute your research interest controls (1, 2). Conversely, a reverse dimerizer lig will bind to dissociate a protein that aggregates in its absence. interest here merize Work? Inducible Homodimerization 동일한 두 단백질의 self-association 유도, or dimerizer, is a cell-permeant organic small molecule with two separate motifs that each bind with odule (Dmr domain) fused onto protein(s) interest. Addition dimerizer brings chimeric 증식Proliferation, 분화differentiation, 접착adhesion, 형질전환transformation, ximity to each or, mimicking activation cellular event that dimerization protein apoptosis 등의 다양한 세포내 과정을 조절하는 in vitro, in vivo 연구에 사용 a reverse dimerizer lig will bind to dissociate a protein that aggregates in its absence. 할 수 있다. B/B Homodimerizer DmrB domain B/B Homodimerizer Inducible Heterodimerization DmrB domain 상이한 두 단백질의 융합 A/C Heterodimerizer DmrA domain 조건부 대립형질을 갖는 수용체receptor나 신호 전달 물질, 또는 다른 두 개의 단백질의 결합에 의해 조절되는 단백질을 만드는 in vitro, in vivo 연구에 사용 할 수 있다. A/C Heterodimerizer DmrC domain DmrA domain DmrC Inducible Reverse domaindimerization 단백질의 분해(가용성solubilization 또는 분리deaggregation) 유도 n DmrD 세포내 domain D/D Solubilizer 분비secretion를 조절하는 in vitro, in vivo 위치location를 조절하거나 연구에 사용할 수 있다. 그림 1. idimerize Inducible Expression의 작동원리 nc. 4 Life Science & Biotechnology 48 1 Clontech Laboratories, Inc. DmrD domain D/D Solubilizer

43 erent dimerization domain recognized by heterodimerizer. Table II: Types Signaling Proteins Activated by idimerize Technology: DmrA Domain DmrC Domain Receptor non-receptor tyrosine kinases Activation Signal Transduction Pathway Receptor non-receptor serine/threonine kinases Non-kinase receptors Signaling proteases Adaptor proteins Example: Inducing a Programmed Cell Death Pathway (Inducible Apoptosis) ntrol Signal Transduction Pathways Activated Caspase Caspase 3 DmrB Domain.1 Apoptosis Figure 1. Fas-induced apoptosis in vitro. The fas receptor (FasR), once trimerized, activates an apoptosis pathway. Programmed cell death can be mimicked at will by transfecting cells with a construct encoding Fas-DmrB fusion protein treating overnight with B/B Homodimerizer to induce trimerization (Panel A). Less than 1 nm B/B Homodimerizer was sufficient to induce maximal cell death in se cells (Panel B; data kindly provided by ARIAD Pharmaceuticals, Inc.). Small Molecule Control Signal Pathways activates Transduction an apoptosis pathway. Programmed cell death can be mimicked at will by 그림 3. Fas-induced apoptosis in vitro. The fas receptor (FasR), once trimerized, Clontech Laboratories, Inc. 2 Activation Signal Transduction Pathway transfecting cells with a construct encoding Fas-DmrB fusion protein treating overnight with B/B Homodimerizer to induce trimerization (Panel A). Less than 1 nm B/B Homodimerizer was sufficient to induce maximal cell death in se cells (Panel B; data B/B Homodimerizer kindly provided by ARIAD Pharmaceuticals, Inc.). Many signaling cascades are A/C activated almost exclusively by inheterodimerizer teractions signaling proteins. Cell surface receptor proteins cluster in response to extracellular factors, which leads to recruitment is ultimately ector protein production, activation or secretion. Any step this signaling pathway can be DmrA brought under dimerizer control by fusing proteins involved to Domain domains recognized bydmrc respective dimerizer lig. 유전자 유도 발현 Heterodimerization 기술을 이용한 idimerize Inducible Expression (Code 63565)은 목적 유전자의 전사 활성을 조절하는데 사용 할 수 있다. DmrB Domain Domain idimerize Inducible Expression 을 이용한 유전자 발현 조절 Activation Inducible Gene Expression ZHFD1 inducible promoter (PZI-1) 하류에 목적Signal 유전자를 클로닝한다. Transduction Pathway Inducible Gene Expression binding component (DmrA/BD fusion; green)가 promoter내에 서열을 인 Th e idimerize Inducible Expression (Cat. No.transcription 63565), anactivation application kit using heterodimerization 식하여 결합한다. 반면 프로모터의 DmrA/-BD가 control transcription activation Heterodimerizer target genes.(cat. No ), an application kit using heterodimerization A/C Th e idimerize Inducible Expression Inducible Gene Expression component(dmrc-ad fusion; red)와 dimerize되어야 전사가 시작되며, 이 control transcription activation target genes. dimerize는 DmrA/-BD와 transcription activation component(dmrc-ad Regulated gene expression using idimerize Inducible Expression. Th e idimerize Inducible Expression (Cat. No ), an application kit using heterodimerization fusion; red)에 대해 친화성을 가진 A/C Heterodimerize에 의해inducible 가능하다.promoter (P ). The binding c Clone yourgene gene interest downstream ZHFD1 control transcription activation target genes. Regulated expression using idimerize Inducible Expression.ZI-1 BD fusion; recognizes binds sequences within promoter. However, activation tranc Clone yourgreen) gene interest downstream ZHFD1 inducible promoter (PZI-1). The binding when DmrA/-BD dimerizes withsequences transcription component (DmrC-AD fusion; BD fusion; green) recognizes binds within activation promoter. However, activation tranr Regulated gene expression using idimerize Inducible Expression. mediated by ir mutualdimerizes affi nity for A/C transcription Heterodimerizer. 세 종류의 element when human-based DmrA/-BD with activation component (DmrC-AD fusion; r your gene interest downstream ZHFD1 inducible promoter (PZI-1). The binding c DmrAClone mediated byutilizes ir mutual affinity for elements: A/C Heterodimerizer. Thefusion; design three human-based BD green) recognizes binds sequences within promoter. However, activation tran is apoptotic signaling cascade can be Domain DmrC 표 2 II: Types Signaling Proteins Activated when DmrA/-BD dimerizes wilements: transcription activation component (DmrC-AD fusion; r The design utilizes three human-based Transcription mimickedtable using idimerize (Figure 1). An in vivo model ( MaFIA Domain activation component: NFkB의 p65 subunit에서 mediated by ir mutual affi nity for A/C Heterodimerizer. Transcription activation component: single 융합되어 DmrC domain, by idimerize Technology: 유래된 transcription activation domaina(ad)이 있는 fused to a transcription ac mouse; 3) utilizes Fas receptor to systematically reversibly Inducible Gene derived Expression from p65 subunit NFkappaB DmrC domain, fused to a transcription ac Transcription activation component: The design utilizes three human-based elements: a single single DmrC domain Activation Receptor non-receptor tyrosine kinases eliminate macrophages from transgenic mice. derived from p65 subunit NFkappaB Receptor non-receptor serine/threonine kinases Signal Transduction Pathway The idimerize Inducible Expression (Cat. No ), an application kit using heterodimerization technology, can be used to control transcription activation target genes. Transcription activation component: singledomains, DmrC domain, fused to a transcription ac binding component: a triplet admra fused to a composite bindin fromcomponent: p65 subunit NFkappaB derived binding 하는 domains 합성 from binding ZFHD1, which consists ZFHD1이라고 two zinc finger Zif268 joined to a homeodoma Non-kinase receptors binding component: triplet DmrA domains, fused to a composite bindin Regulated gene expression using idimerize Inducible Expressiona. domain which (BD)이 융합되어 있는 DmrA triplet. ZFHD1 Clone your gene interest downstream ZHFD1 inducible promoter (PZI-1).domains의 domains binding component (DmrA/ZFHD1, consists two zinc fiThe nger from Zif268 joined to a homeodoma BD fusion; green) recognizes 은 binds sequences within promoter. However, activation transcription only occurs Oct-1의 homeodomain에 결합하는 Zif268의 2개의 zinc Inducible promoter component (PZI-1 ):DmrA ZFHD1 binds nity specifi to when DmrA/-BD dimerizes with binding transcription activation component (DmrC-AD fusion; red) with at fused high promoter, component: a triplet domains, toaffi a composite city bindin finger domain으로 있다. sequence, but not to Zif268 or Oct-1 binding sites (4). T mediated by ir mutual affinitycomposite for A/C Heterodimerizer. ZHFD1구성되어 binding Signaling proteases Adaptor 1 proteins which consists two zinc finger domains joined to a specifi homeodoma ZFHD1, Inducible promoter component (PZI-1 ): ZFHD1 bindsfrom withzif268 high affi nity city to a binding minimalsequence, promoter but derived from PIL2.or Oct-1 binding sites (4). T composite ZHFD1 not to Zif268 placed downsteam a minimal promoter derived from PIL2. Inducible promoter component (P(P ):):azhfd1는 ZHFD1 Transcription activation component: a single DmrC domain, fused transcription activation domain (AD) Inducible promoter component ZFHD1 binds with high affinity specificity to ZI-1to ZI-1 derived from p65 subunit NFkappaB Activation composite ZHFD1 binding but notdomain to Zif268 binding sequence에 높은Transcription 특이성과sequence, 친화성으로 결합한다. 그러 or Oct-1 binding sites (4). T DmrC Viability (% control) placed downsteam The design utilizes three human-based elements: 8 Example: a Programmed Death Pathway 실험 예 : Inducing 프로그램화된 세포 사멸 경로의Cell 유도(Apoptosis 유도) (Inducible Apoptosis) 6receptor (FasR) is a transmembrane protein located on e Fas surface cells(fasr)는 that activates programmed death (apoptosis) Fas receptor apoptosis를 활성화 cell 시키는 세포 표면에 위치한 when induced to trimerize by fas lig (FasL) located transmembrane protein으로 인접한 세포(예, 세포 독성 T on 세포)표면 4 surface adjacent cells (e.g., cytotoxic T cells). FasL/FasR binding 의 fas lig (FasL)가 trimerize될 때 apoptosis가 유도된다. FasL/ plays an important role in regulation immune system is apoptotic signaling cascade can be Transcription Activation Domain mimicked using idimerize in vivo model ( MaFIA DmrC (Figure 1). An Inducible promoter component (P ): ZFHD1 binds with high affinity A/C specifiheterodimerizer city to 12 repeats a unique Domain DmrA/ mouse; 3) utilizes Fas receptor to systematically reversibly composite ZHFD1 binding sequence, but not to Zif268 or Oct-1 binding sites (4). The binding sites are -BD A/C Heterodimerizer placed downsteam a minimal promoter derived from P. DmrA/ eliminate macrophages from transgenic mice. fusion ZI-1 IL2 FasR 반응은 면역 시스템과 암의 진행 과정을 조절하는데 중요한 역할 2 을 한다. 이 apoptosis 시그널 과정은 idimerize(그림 3)를 이용해 재현 A 할 수 있다. 생체 모델( MaFIA mouse; 3)은 Fas receptor를 이용 Fas death domain.1 형질전환 가역적으로 1 에서 하여 체계적이고 쥐transgenic mouse 마크로파지 DmrA/ -BD 1 fusion SEAP Activity (RU) 4 Apoptosis -BD fusion B Death-inducing signaling complex (DISC) Caspase 3 -BD fusion DmrA/ Transcription Activation Domain DmrC Domain (membrane-anchored) Caspase 8 (4) from P. placed downsteam a Transcription minimal derived Activation Domain Domain. 이 결합IL2 나 Zif268나 site에는promoter 결합하지 않는다 DmrC Oct-1 융합하는 binding component: a triplet DmrA domains, fused to a composite binding domain (BD) called 에서 유래된 minimal promoter의 위치한다. site는 PIL2 ZFHD1, which consists Domain two zinc finger domains from Zif268 joined to a하류에 homeodomain from Oct-1 를 제거한다. FasR), once trimerized, activates an apoptosis pathway. Programmed cell death can be mimicked at will macrophages mrb fusion protein treating overnight with B/B Homodimerizer to induce trimerization (Panel A). Less aximal cell death in se cells (Panel B; data kindly provided by ARIAD Pharmaceuticals, Inc.). lontech.com 1 Viability (% control) Death-inducing signaling complex (DISC) B (membrane-anchored) B Fas death domain (membrane-anchored) 다른 두 개의 단백질을 dimerization 시킨다. Fas death domain A B/B Homodimerizer e idimerize Inducible Homodimer (Cat. No ) Activation Signal Transduction uses B/B Homodimerizer lig, which incorporates twopathway identises cal binding motifs, to induce self-association a single signaling e idimerize Inducible 그림 2. idimerize Inducible s Heterodimer (Cat. No ) uses A/C Heterodierent binding motifs, to erent proteins interest,2개의 each 다른 idimerize Inducible Heterodimer (Code 63567)은 erent dimerization domain recognized by binding motif를 가진 A/C Heterodimerizer lig를 이용하여 각기 h Pathway (Inducible heterodimerizer. Apoptosis) ein located on ath (apoptosis) L) located on asl/fasr binding mmune system is apoptotic signaling cascade can be mimicked using idimerize (Figure 1). An in vivo model ( MaFIA mouse; 3) utilizes Fas receptor to systematically reversibly eliminate macrophages from transgenic mice A/C Heterodimerizer Inducible Promoter 8 4 Transcription Inducible Promoter Inducible Promoter Concentration 1.1 (nm)1 A/C Heterodimerizer 1 (AP21967) Concentration (nm).1 A/C Heterodimerizer (AP21967) Figure 2. Dose-dependent control gene expression with idimerize Inducible Expression. HT18 Tran Tran Tran A/C Heterodimerizer Inducible Promoter 4 SEAP Activity SEAP SEAP Activity (RU) Activity (RU)(RU) Cat. No ) porates two identisingle signaling ze Inducible e A/C Heterodiding motifs, to f interest, each recognized by e Fas receptor (FasR) is a transmembrane protein located on surface cells that activates programmed cell death (apoptosis) when induced to trimerize by fas lig (FasL) located on surface adjacent cells (e.g., cytotoxic T cells). FasL/FasR binding plays an important role in regulation immune system Viability (% control) usively by inptor proteins cluster o recruitment is ultimately production, pathway can be oteins involved to g. Mammalian Inducible Expression 1

44 aggregation is resting state, D/D Solubilizer breaks up is version technology can be used for rapid, reversible changes in subcellular location, aggregation state /or biological activity engineered proteins. Transcription Activation Domain A/C Heterodimerizer DmrA/ -BD fusion Mammalian Inducible Expression An innovative application this technology is inducible protein e D/D Solubilizer lig can be Transcription added to induce protein secretion from cells in a matter 15 minis method has been used to induce rapid, transient, tightly-regulated secretion insulin in a mouse model for diabetes. Induced release insulin reversed hyperglycemia (5). Inducible Promoter SEAP Activity (RU) 4 A B No Lig With Solubilizer Lig ER Golgi 1 Concentration (nm) A/C Heterodimerizer (AP21967) Figure 2. Dose-dependent control gene expression with idimerize Inducible Expression. HT18 그림 4.cells Dose-dependent control gene expression with (SEAP) idimerize were stably transfected with secreted alkaline phosphatase reporterinducible gene DmrC-AD/ DmrA/-BD treated increasing concentrations A/C Heterodimerizer. Expression.constructs, HT18 cells were with stably transfected with secreted alkalinein absence A/C Heterodimerizer, target gene expression was undetectable. Half-maximal induction occurred at 2 nm phosphatase (SEAP) reporter gene DmrC-AD/DmrA/-BD constructs, A/C Heterodimerizer. treateddata withkindly increasing A/C Heterodimerizer. In absence A/ suppliedconcentrations by ARIAD Pharmaceuticals Inc. C Heterodimerizer, target gene expression was undetectable. Half-maximal induction 3 Clontech Laboratories, Inc. occurred at 2 nm A/C Heterodimerizer. Supernatant luciferase activity (RLU) 템은 당뇨병 연구를 위한 마우스 모델에서 신속하고 일시적이며 정밀 Protein Secretion 고혈당증hyperglycemia을 완화시킨다. 한 인슐린 분비를 유도하는데 이용하고 있다. 인슐린 분비를 유도하면 m (Cat. No ) olubilizer breaks up e technology can be ular location, aggreered proteins. inducible protein bilizer lig can be a matter 15 mininduce rapid, tranin a mouse model d hyperglycemia (5). DmrD reverse dimerization domains D/D Solubilizer 4 Your protein DmrD Domain 45, 4, 35, 3, 25, 2, 15, 1, 5, ConcentrationInc. D/D Solubilizer (nm) Clontech Laboratories, 그림 6. Secretion DmrD-tagged luciferase after addition D/D Solubilizer. 7 hr after transfection with a DmrD-tagged Metridia luciferase construct, cells were split Figure 4. Secretion DmrD-tagged luciferase after addition D/D Solubilizer. Plasma m Inactive or aggregated protein e m aa nective or monomeric protein into wells a transfection 6-well plate.with Thea DmrD-tagged medium wasmetridia removedluciferase fresh mediumcells was added 7 hr after construct, br D/D Solubilizer Outside cell were split into wells a 6-well plate. The Solubilizer. medium was18removed mecontaining increasing concentrations D/D hr later, fresh media was coldium was added containing increasing concentrations D/D Solubilizer. 18 hr lectedlater, analyzed using Clontech s Ready-To-Glow Secreted Luciferase Reporter media was collected analyzed using Clontech s Ready-To-Glow Figure 3. The idimerize Reverse Dimerization enables dose-dependent (Code Luciferase ). Reporter (Cat. No ). Secreted 5, 45, control protein secretion. Fusion proteins containing DmrD domains localize to endoplasmic reticulum as aggregates (Panel A). When D/D Solubilizer is added, it dissolves aggregates allows protein to be exported through secretory apparatus (Panel B). 4, 35, 3, 25, 2, 15, 42 Life Science & Biotechnology 48 1, 5, Concentration D/D Solubilizer (nm) DmrD 5, Furin cleavage site Protein interest Supernatant luciferase activity (RLU) Solubilizer Lig Golgi 5, 45, 4, 35, 3, 25, 2, 15, 1, 5, Furin cleavage site In idimerize Reverse Dimerization (Cat. No ) Protein aggregation is resting state, D/D Solubilizer breaks up interest Plasma m DmrD e m ane r b is version technology can be reverse D/D 단백질 분비의 일시적 조절 Solubilizer dimerization Outside cell D/D Solubilizer used for rapid, reversible changes in subcellular location, aggredomains 그림 5. The idimerize Reverse Dimerization enables dose-dependent control gation state /or biological activity engineered proteins. Figure 3. The idimerize Reverse Dimerization enables dose-dependent idimerize Reverse Dimerization (Code 63566)은 휴면 상태일 protein secretion. Fusion proteins containing DmrD domains localize to enan innovative application this technology is inducible protein control protein secretion. Fusion proteins containing DmrD domains localize Yourreticulum as aggregates (Panel A). When D/D Solubilizer is added, it 때 결합이 일어나고 D/D Solubilizer가 첨가되면 eprotein-protein 상 doplasmic D/D Solubilizer lig can be to endoplasmic reticulum as aggregates (Panel A). When D/D Solubilizer protein aggregates allows protein to be exported secretory is added, it dissolves aggregates allows protein tothrough be exported added induce protein secretion from cells in a matter 15 min-dissolves 호 결합이 분리된다. 이 to 시스템을 이용하면 단백질의 응집상태나 생물 through secretory apparatus (Panel B). (Panel B). is method has been used to induce rapid, tran- apparatus 학적 활성 그리고 세포 내 위치subcellular location연구에서 신속하고 가역 Inactive or aggregated protein Active or monomeric protein sient, tightly-regulated secretion insulin in a mouse model 적인 변화에 이용할for수diabetes. 있다. Induced release insulin reversed hyperglycemia (5). 15분 만에 세포로부터 단백질 분비를 유도할 수 있다(그림 6). 이 시스 Inactive or aggregate DmrD Domain (Data kindly supplied by ARIAD Pharmaceuticals Inc.) 실험inducible protein secretion 이 있다. D/D Solubilizer lig를 첨가하면 ER Your protein Con Temporal Control Protein Secretion B 단백질 이 기술의 혁신적인A적용 예로는 그림 5에서 설명한 분비 유도 No Lig With Solubilizer Lig DmrD reverse dimerization domains Supernatant luciferase activity (RLU) DmrC Domain Figure 4. Secretion Dmr 7 hr after transfection with were split into wells a 6dium was added containin later, media was collec Secreted Luciferase Report

45 Mammalian Inducible Expression 제품명 ( 구 ARGENT Kit 제품명 ) Size Code Vector s (include 5 μl aliquot dimerizer lig) idimerize Inducible Homodimer (ARGENT Regulated Homodimerization Kit) idimerize Inducible Heterodimer each (ARGENT Regulated Heterodimerization Kit) idimerize Reverse Dimerization each (RPD Regulated secretion / Aggregation Kit) idimerize Inducible Expression each (ARGENT Regulated Transcription Plasmid Kit) Dimerizer Ligs (in vitro format supplied at.5 mm in ethanol) each μl 6356 B/B Homodimerizer (AP2187) 5 x 5 μl μl A/C Heterodimerizer (AP21967) 5 x 5 μl μl D/D Solubilizer* (AP21998) 5 x 5 μl Dimerizer Ligs (in vivo format supplied dry) B/B Homodimerizer (AP2187) 5 mg A/C Heterodimerizer (AP21867) 5 mg D/D Solubilizer* (AP21998) 5 mg 주문생산 *D/D Solubilizer 는 AP21998 과다른물질이지만동등한기능을한다. References 1.. Clackson, T. et al. (1998) Redesigning an FKBP-lig interface to generate chemical dimerizers with novel specifi city. Proc. Natl. Acad. Sci. USA 95(18): Clackson, T. (27) In Chemical Biology: From Small Molecules to s Biology Drug Design, Eds. Schreiber, S. L. et al. (Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim, Germany), pp Burnett, S. H. et al. (24) Conditional macrophage ablation in transgenic mice expressing a Fas-based suicide gene. J. Leukoc. Biol. 75(4): Pomerantz, J. L. et al. (1995) Structure-based design transcription factors. Science 267(5194): Rivera, V. M. et al. (2) Regulation protein secretion through controlled aggregation in endoplasmic reticulum. Science 287(5454): License Notice [K73] 43

46 Mammalian Inducible Expression Inducible Expression Low background, High Sensitivity 의 3 세대 Tetracycline Inducible Gene Expression s은가장강력하고적용성이뛰어난 3세대포유류유도발현시스템으로정밀하게유전자발현을조절할수있도록기존제품의성능을개선하였다. 본시스템을이용하면좀더가역적이고정량적이며재현성있게목적유전자의발현을조절할수있다. Hermann Bujard와 Manfred Gossen, Wolfgang Hillen 연구그룹 (1,2) 에의해개발된 시스템은 promoter 변형으로백그라운드를현저하게줄이고 transactivator protein 개선을통해 Dox에대한민감도를한층높인혁신적인 Tet 시스템이다. Inducible 의개요 transactivator protein을발현하면서 TRE promoter (P TRE ) 로목적유전자를조절하는 세포는독시사이클린 doxycycline (Dox; tetracycline analog) 이첨가되었을때목적유전자를고발현시킨다. protein은 Dox가결합하면형태적인변화가생겨 P TRE 내의 tet operator (tet O) 에결합하게된다 ( 그림 1). 새로운시스템은기존의낮은수준의 TetR-based system (Clontech 에서는판매하지않는제품 ) 과는달리, 기술을적용하여 transcription을저해하기보다는촉진시키는것으로극도로낮은기저발현basal expression과높은목적유전자발현이가능하다. 뿐만아니라한층더빨라진반응시간등의장점을갖고있으며특히 in vivo 실험에최적이다. No Dox P TRE Promoter Transactivator 극도로낮은백그라운드와높은감도 시스템은최적화된두가지요소의결합으로기존의 Tet- On 시스템보다한층더성능이향상되었다. P TRE promoter - 기존의 inducible promoter의변이형으로최대 2 배가량백그라운드발현을줄였다 ( 그림 2). transactivator protein - 유도물질인 Dox에대한감도를획기적으로높여아주낮은농도의 Dox (5~1 ng/ ml ) 에서도기존 Advanced 보다 1~15 배이상발현배율을증가시켰다 ( 그림 3). 두가지요소를조합하면 1 ng/ ml의 Dox에서도목적유전자를높은수준으로발현시킬수있을뿐만아니라 ( 그림 3), 백그라운드발현이매우낮아 transient cotransfection을통해서도발현유도시점과발현을유도하지않은시점에서의발현량이 27, 배이상차이가나는것을확인하였다 (MCF cells, data not show). 그림 2. The P TRE promoter results in significantly 16, 8 Advanced reduced basal expression. 12, Reduced 6 Background HEK 293 cells were transiently cotransfected with 8, both response vectors (containing luciferase) regulator vectors from each 4, Tet- On Advanced Inducible Expression s. The 5 5 ng/ml cells were cultured in presence absence Dox, after 24 hr, luciferase expression was measured. Although both systems provided strong expression in presence Dox, produces far lower background expression in absence Dox (inset). RLU Dox Doxycycline Transcription 그림 1. The systems allow inducible gene expression only in presence doxycycline. When Dox binds, transactivator undergoes a conformational change allowing it to bind tet operator (tet O) repeats within TREG Promoter (P TRE ). The transactivator activates expression through transcription activation domain repeats. A kd 7 5 B RLU 16, 12, 8, 4, Dox (ng/ml) ptre-luc 1 ¹ 1 ng/ml 1¹ 1² 1³ Luciferase b-actin 그림 3. is highly sensitive to as little as 1 ng/ ml doxycycline. Following cotransient transfection pcmv- Tet P TRE -Luc in HeLa cells, increasing levels Dox were added expression luciferase was measured using an antiluciferase antibody (Panel A) a luciferase assay (Panel B). Induced expression was very high even with Dox concentrations as low as 1 ng/ ml, was detectable even at.1 ng/ ml Dox. 44 Life Science & Biotechnology 48

47 Mammalian Inducible Expression - 두 개의 최적화된 요소의 강력한 조합 다양한 Vector Clontech에서는 다양한 형태의 vector가 포함된 시스템을 판매하고 있으며, 각 시스템에는 아래 구성품도 포함되어 있다. (1) Transactivator Protein 는 Advanced transactivator protein의 성능을 Te continued Choice Vect conti 개선하여 독시사이클린doxycycline에 대한 감도를 높였다(4). transactivator vector (pcmv-tet 또는 pef1α-tet) 변형된 박테리아 Tet repressor (TetR)를 3 Transactivator Protein은 continued continued continued continued PTRE promoter와 multiple cloning site가 포함된 TRE response vector continued 개의 minimal VP16 activation domain에 융합하여 새로운 전사 활성 Figure&7 puromycin shows range markers vector formats available. Each is sold as a complet Hygromycin linear selection Choice Vector Formats Figure 7 shows range vector format a (eir pcmv-tet Choice transactivator Vectorvector Formats The The The The is a을 Powerful is a Powerful is이combination a단백질은 is Powerful acombination Powerful Combination 단백질 만들었다. 기존의Combination Advanced transactivator The ismutation a Powerful Combination Two Elements Two Elements Two Optimized Two Elements Optimized Elements by Optimized Optimized by Mutation by Mutation by Mutation transactivator 에서 5 개의by아미노산을 변형시킨 것이다 ( Two Elements Optimized Mutation Tet Approved FBStransactivator vector (eir pcmv-tet or pef1a-tet) a Choice Vector For Figure 7 shows reagent range with promoter vector formats available. Eachsite is sold as a co a Pa TRE response vector with acloning PTRE promoter Xfect transfection a TRE response vectorfigure a multiple 7 shows TRE range vector formats available. E continued Transactivator Transactivator Protein Transactivator Protein Transactivator Protein transactivator 서열Protein 비교는 사이트 참조). Tet- a transactivator vector pcmv-tet pef1a-tet) (eir both hygromycin or puromycin linear selection m Transactivator both hygromycin puromycin linear selection markers The Advanced a Powerful Combination is a modified is aform modified anform isimproved a modified Protein isana improved modified form form an improved transactivator Advanced an is improved transactivator Advanced protein Advanced which transactivator protein hastransactivator which been evolved protein has been to protein which evolved display has which far to been display higher has evolved been far evolved higher to display to display far higher far higher a transactivator vector (eir pcmv-tet or pef1a-t a modified form an(4). Advanced protein which hasminimal been evolved to display higherminimal (1) Transactivator On 는 기존 아주 높은 나타내며, 낮은 Dox 농far continued Two Optimized by Mutation sensitivity to sensitivity doxycycline to doxycycline sensitivity (4).is The sensitivity protein to(4). doxycycline The consists toelements protein doxycycline improved consists a버전에 The modified (4). protein The a비해 bacterial modified protein consists Tet consists bacterial repressor atransactivator modified Tet a 발현율을 (TetR) modified repressor bacterial fused bacterial (TetR) Tet to repressor three fused Tet repressor to(tetr) three VP16 fused (TetR) minimal activation to fused three VP16 tominimal activation three VP16 activation VP16 activation Vectors sensitivity to doxycycline (4). The protein consists a modified bacterial Tet repressor (TetR) fused to three minimal VP16 activation a TRE response vector with a P promoter a multiple cloning Tet Approved FBS Tet Approved FBS a TRE response vector with a P domains todomains create a to transcriptional create domains a transcriptional domains toactivator createtoacreate protein transcriptional activator a transcriptional (transactivator). protein activator (transactivator). activator Five protein amino protein (transactivator). Five acidamino (transactivator). changes acid Five convert changes amino Five Clontech s convert acid amino changes Clontech s acid changes convert Advanced convert Clontech s Advanced Clontech s Advanced Advanced promoter asite multiple clo 도에도 민감하게 반응한다. 이와 같이 Dox에 대한 민감도가 증가함으 protein을 발현 TRE domains to create a transcriptional activator protein (transactivator). Five amino acid changes convert Clontech s Advanced pcmv-tet는 CMV promoter로부터 TRE Transactivator Protein transactivator transactivator to transactivator to (fortransactivator a comparison to (for a comparison to (for various a comparison (for various a comparison transactivator various transactivator sequences, various transactivator sequences, see transactivator see sequences, sequences, see see is a modified form an improved protein which has see beenwww.clontech.com). evolved to display far higher transactivator to (for a comparison variousadvanced transactivator transactivator sequences, Xfect transfection reagent vector로 both hygromycin 제외한 puromycin linear selection markers Xfect transfection reagent generates very generates high maximal verygenerates high maximal generates very high expression maximal responds high maximal expression tothe responds lower expression Dox to lower concentrations responds concentrations to concentrations lower than todox its lower predecessors. concentrations than Dox its concentrations These than Dox its형질전환연구 than These predecessors. concentrations its Dox predecessors. concentrations These Dox These concentrations Dox concentrations both 모든 hygromycin linear 로써 낮은 농도로 투여할 수 있기 때문에 세포 배양이나 시키는 EF1α 버전을 puromycin 에 포 selection markers sensitivity to very doxycycline (4). protein consists a Dox modified bacterial Tet repressor (TetR) fused to three minimal VP16 activation generates veryexpression high maximal expression responds toresponds lower Dox than itspredecessors. predecessors. These Dox concentrations domains to create a transcriptional activator protein (transactivator). Five amino acid studies. changes convert Clontech s are far below arecytotoxic far are below cytotoxic are far for below are eir levels farcytotoxic below cell for culture eir cytotoxic levels cell or transgenic culture for levels eir for or cell transgenic studies. eir culture cell The studies. culture orincreased transgenic or The transgenic Dox increased studies. sensitivity The Dox increased is sensitivity particularly The increased Dox isis particularly sensitivity advantageous Dox Advanced sensitivity isadvantageous particularly is particularly advantageous advantageous far levels below cytotoxic levels for eir cell culture or transgenic studies. The increased Dox sensitivity particularly advantageous transactivator tostudy 시 (for a comparison 줄일 various transactivator sequences, see Inducible Expression (EF1α version) 함되어 있다. 세포 독성을 수 있다. 특히, 높은 Dox 농도를 유지 transgenic Tet Approved FBS available. Each is sold as a complete system for in vivo for studies in vivo inin tissues studies forstudies where ininvivo tissues for high studies intissues vivo where Doxin studies concentrations high tissues Dox in where tissues concentrations high where are difficult Dox high concentrations are Dox todifficult attain concentrations (e.g., toare attain brain). difficult (e.g., are difficult to brain). attain to(e.g., attainbrain). (e.g., brain). Tet Approved FBS Figure 7 shows range vector formats for vivo in where high Dox concentrations difficult to attain (e.g., brain). generates very high maximal expression responds toare lower Dox concentrations than its predecessors. These Dox concentrations Choice Vector Formats Transactivator (Code )에는 EF-1 αvectors promoter로부터 transactivator를 발현하 Transactivator (eir Xfect transfection reagent a transactivator vector pcmv-tet orvectors pef1a-tet) Xfect transfection reagent pcmv-tet expresses protein constitutively from a CMV promoter, is in 는 vector가 포함되어 있다. Table I: Three TableGenerations I: Three Table Generations I:Table ThreeI:Generations Inducible Three Generations Inducible Expression Expression s Inducible Inducible s Expression Expression s s 표 1 I: Three Table Generations Inducible Expression s pcmv-tet expresses protein constitut except EF1 alpha version, in which an EF-1 alpha promoter expresses transactivator a TRE response vector with a P promoter a multiple cloning site Table I: Three Generations Inducible Expression s TRE Generation Transactivator Protein Inducible Generation Generation Transactivator Protein Transactivator Inducible ProteinProtein Inducible PromoterPromoter Promoter Inducible Inducible Promoter Promoter Name NameName Name Name Generation Generation Transactivator Protein Transactivator except Vectors EF1 alpha version, in which an EF-1 alpha p Transactivator Name Generation Transactivator Protein Inducible Promoter PP 1st 1st 1st 1st 1st P P P pcmv-tet both hygromycin puromycin linear selection markers Transactivator Vectors 1st P 하기 어려운 조직(뇌 등)의 in vivo 실험에 유용하다. are far below cytotoxic levels for eir cell culture or transgenic studies. The increased Dox sensitivity is particularly advantageous for in vivo studies in tissues where high Dox concentrations are difficult to attain (e.g., brain). TRE2 TRE2 TRE2 2nd2nd 3rd 3rd 3rd 3rd 3rd TRE2 Advanced 2nd Advanced Advanced Advanced Advanced PTIGHT Advanced P 3rd TIGHT PTRE PTRE TRE2 TRE2 PPTIGHT PTIGHT PTIGHT PPTRE PTRE TIGHT TRE pcmv-tet expresses protein constitutively from a C pcmv-tet r constitutively from a CMV promoter, PCMV IEFBS PSV4 pcmv-tet expresses Neoversion, P Tet Approved except EF1protein alpha in which an EF-1 alpha promoter expre TRE PCMV P promoter except EF1 alpha version, in which anie EF-1 alpha expresses r transacti Neo SV4 Xfect transfection reagent pef1a-tet HEK 293 Cells HeLa Cells HEK 293 1,6 Cells HEK 293 Cells HEK 293HEK Cells 293 Cells 1,6 1,6 1,6 1,6 8, 8, 8, 1,6 8, 1,2 HeLa Cells HeLa Cells HeLa Cells 8, pcmv-tet Advanced Advanced 8 8 Fold induction Fold induction 8 1,2 8 Fold inductionfold induction 1,2 1, , 1, , 4, 4, 4, 6, 6, 4, 4, 1st 2nd Generation 3rd 2, 2, 2, Neor P Transactivator Vectors PSV4 PEF-1aNeor pef1a-tet CMV IE PSV4 Neor pcmv-tet expresses protein constitutively from a CMV promoter, is included i r PEF-1a P 그림 5. EF1 Transactivator Vectors promoter Neo Response Vectors pef1a-tet SV4 except alpha version, in which an EF-1 alpha expresses transactivator. All response vectors contain a PTRE promoter. ptre is included in both core system r 1st 2, 2, 2nd Generation PSV4 Response Vectors Neo 모든 response vector는 P TRE promoter를 포함하고 는 ptre-ires can있으며 inducibly coexpress any two genes All response vectors contain a P P TRE promoter. ptre is included Figure 5. Comparison three generations. Panel A shows transactivator inducible promoter combinations for each generation system. was launched by Clontech in 1996 at time this was premier inducible expression system, its performance has only been surpassed by subsequent generations system. Panel B shows co-transient transfection experiments with both vectors in HEK 293 HeLa cells. Although comparing difference between induced 1st when2nd 3rd 1st 2nd Advanced shows 3rdgreat improvement over original uninducedgeneration states (fold induction), shows even higher fold induction due to significantly reduced basal expression provided by Generation promoter. P TRE 1st 2nd 1st2nd 1st 3rd 2nd3rd 2nd 3rd 2nd 1st 2nd 1st 3rd 2nd3rd 2nd 3rd 3rd 1st TRE ptre 3rd 그림 4. Comparison three generations. Panel A shows transacti- PSV4 pcmv-tetpef-1a 2, pef1a-tet PCMV PSV4 Neor IE ptre-ires can inducibly coexpress any two genes interest, is included with (2) Response Vectors Response Vectors r response vectors a PTRE promoter. Alternatively, you inducibility using red orcontain green fluorescent proteins if ptre3 you are PCMV PSV4 All can monitor Neo IE 4, 4 6, pcmv-tet Advanced Advanced Advanced Advanced 6, 1,2 PEF-1a HeLa Cells HeLa Cells 8, pef1a-tet ptre-ires can inducibly coexpress any two genes using interest, a Response Vectors Alternatively, you can monitor inducibility red or Inducible Expression (Code )과 MCS HEK 293 Cells Fold induction Fold induction Advanced Advanced Advanced Advanced Advanced 2nd 2nd 2nd Advanced P Alternatively, you can monitor inducibility using red or green fluores r pa PSV4 (EF1α Neo EF-1a TRE AllPresponse vectors contain a ptre PTRE promoter. ptre is included in both core s Inducible Expression Version) (Code )의 ptre-ires can inducibly coexpress interest, is included wit ptre ptre-ires 구성품이다. 두 종류의 유전자를 함께 발현시킬any 수 two 있는genes PTRE-IRES는 3rd PTRE pa red or green fluorescent proteins if yo Alternatively, you can monitor inducibility using P 1st 1st MCS vator inducible promoter combinations for each generation system. was launched by Clontech in 1996 at time this was premier inducible bicistronic IRES system에 P Vectors pa 포함되어 있다. 목적에 따라 적색 또 Response expression system, its performance has only been surpassed by subsequent gen는 녹색 형광단백질이 포함된 mcherry 또는 ZsGreen1 시스템을 선택 both core system th erations system. Panel B shows co-transient transfection experiments is included with bicistro 할 수도 Pby P with both vectors in HEK 293 HeLa cells. Although Advanced shows great uninduced states uninduced (fold induction), states uninduced (fold induction), uninduced states shows (fold states induction), even (fold shows higher induction), even fold induction higher shows fold due even induction shows to higher even significantly due fold higher toinduction fold significantly reduced induction duebasal to reduced due expression significantly to basal significantly provided expression reduced bybasal reduced provided expression basal by expression provided provided by 있다. pa P IRES pa TRE IRESred orpa TRE PTREyoumCherry Alternatively, can monitor inducibility using green fluorescent proteins if you are using t IRES pa TRE promoter. promoter. promoter. promoter. P P P P improvement over original when comparing difference between ptre-mcherry ptre-ires ptre-mcherry ptre induced uninduced states (fold induction), shows even higher fold ptre-zsgreen1 이를 tetracycline response element (TRE)라 부른다. tet operator 반복 서열은 모든 시 스템에서 공통적으로 사용되었지만 PTRE의 연결 부위 서열의 간격과 중 앙 부분의 배열이 변경되었고 minimal CMV promoter 의 요소도 일부 변형되어 있다. PTRE는 내인성 포유류 전사 요소가 결합 할 수 있는 부 위가 적어졌고 transactivator가 없을 때는 비활성화되기 때문에 어느 TRE 계열의 promoter보다도 낮은 기저발현basal expression을 나타낸다. 결과적으로 PTRE promoter와 transactivator의 결합으로 기존의 포유류 유도 발현 시스템보다 더 높은 유도 발현율을 보일 수 있게 되었다. MCS MCS MCS MCS P IRES TRE ZsGreen1 Markers IRES pap ZsGreen1 MCS pa IRES pa for inducible clones can be performed using eir hygromycin or puromycin line ptre-zsgreen1 그림 6. Response Vectors Both markers are included with every system. 3P Linear Selection Markers mcherry IRES pa TRE PLinear Hygromycin MarkerIRES Linear Selection Markers pafor inducible Selection clones can be performed using eir hygrom TRE ZsGreen ptre-zsgreen1 Both markers are included with performed system. r Selection for inducibleevery clones can be using PSV4 Markers Hyg (3) Linear Selection pa PTRE ZsGreen1 IRES pa Both markers are included with every system Linear Hygromycin Marker MCS Selection ptre-mcherry TRE MCS 시 발현율을 최대로 끌어올린다(3). CMV promoter의 상류에 19 bp의 pa ptre-zsgreen1 PTRE mcherry Clontech Laboratories, Inc. pa IRES ptre-zsgreen1 PTRE IRES pa Linear Selection PTRE promoter는 기저 발현basal expression을 극도로 억제해서 발현 유도 PTRE P mcherry IRES pa TRE mcherry MCS pa IRES MCS IRES ZsGreen1 TRE MCS MCS MCS PTRE PTRE P pa ptre-ires ptre-mcherry (2) PTRE Inducible Promoter ClontechClontech Laboratories, Laboratories, Clontech Inc.tet Clontech Laboratories, Inc. Laboratories, Inc. Inc. operator 서열이 7개 반복되어 있는데, 3 MCS moter. MCS Clontech Laboratories, Inc. MCS induction due to significantly reduced basal expression provided by PTRE pro- MCS TRE MCS TRE MCS TRE MCS TRE MCS MCS MCS MCS Figure 5. Comparison Generation three generations. Panel A shows transactivator inducible promoter combinations for each generation Generation Generation Generation Generation Generation Generation Generation pa system. was launched by Clontech in 1996 at time this was premier inducible expression system, its performance has TRE TRE Figure 5. Comparison Figure 5. Comparison Figure generations 5. Figure Comparison three 5. generations Comparison. three Panel. generations A shows three Panel generations A transactivator shows. Panel. transactivator AB shows Panel inducible A shows promoter transactivator inducible transfection transactivator combinations promoter inducible combinations for inducible each promoter generation for promoter combinations each generation combinations for 293 each generation for each generation only beenthree surpassed by subsequent generations system. Panel shows co-transient experiments with both vectors in HEK HeLa cells. Although Advanced shows great improvement over was comparing difference between induced system. system. was launched was system. by launched Clontech system. by in was 1996 at Clontech launched was in 1996 at launched time by Clontech this was by time Clontech in 1996 at this premier inoriginal 1996 at inducible time premier this expression time was inducible this when premier was system, expression inducible premier system, its performance inducible expression its expression performance system, has system, its hasperformance its performance has has ptre ptre-ires All response vectors contain a ptre-ires PTRE promoter. ptre is included in uninduced states (foldsurpassed induction), shows even higher induction due B to shows significantly reduced basal expression provided by only been surpassed only been bysurpassed subsequent only been by generations subsequent only surpassed been generations by subsequent by subsequent system. generations Panel generations system. Bshows Panel co-transient fold B system. shows co-transient transfection Panel system. Panel transfection experiments B co-transient shows experiments co-transient with transfection both vectors transfection with experiments both in HEK vectors experiments 293with in HEK bothwith 293 vectors bothin vectors HEK 293 in HEK 293 ptre-mcherry promoter. cells. PTRE HeLa cells. Although HeLa Although Advanced HeLa cells. HeLa shows Although Advanced cells. great Although shows improvement Advanced great improvement Advanced over shows great original shows over improvement great original improvement over when over original comparing when original comparing difference when between difference comparing when comparing between induced difference induced difference between between induced induced can inducibly coexpress any two genes interest, ptre-ires Linear selection marker인 hygromycin 또는 puromycin을 사용하여 Linear Puromycin Marker Linear Selection Markers r PSV4 pa clone을 선별할 수 있으며r 두 marker 모두 system에 포함 LinearHyg Hygromycin Marker PSV4for inducible Puro pa Selection clones can be performed using eir hygromycin or puromyci Linear Selection Markers 되어 있다. r Linear Puromycin Marker PSV4 Hyg Both markers are included with every system. pa Selection for inducible clones can be performed using eir hygromycin or puromycin linear select r PSV4 Puro pa Both markers are included system. Linear Hygromycin Marker with every Linear Puromycin Marker Figure 7. Vector formats for systems. For complete components lists, see Linear Hygromycin Markerr PSV4 PSV4 Hyg Hygr pa PSV4 Puror pa Figure 7. Vector formats for systems. For complete components list pa Linear Puromycin Marker Clontech Laboratories, Inc. Puromycin Marker 그림7.Linear Linear Selection Markers r PSV4 PSV4 Puro Puror pa pa Figure 7. Vector formats for systems. For complete c Clontech Laboratories, Inc Figure 7. Vector formats for systems. For complete components lists, see Figure 7. Vector formats for systems. For complete components lists, see Clontech Laboratories, Inc. Clontech Laboratories, Inc. Inc. Clontech Laboratories,

48 Mammalian Inducible Expression 조혈모세포와줄기세포에서의유도발현예 Inducible Expression (EF1α Version) 은 EF-1 alpha promoter가포함된 버전으로이를이용하면시간이지남에따라 CMV promoter에서유전자 silencing 현상이나타난다고알려진줄기세포나조혈모세포등에서도 transactivator를장기간안정적으로발현시킬수있다 ( 그림 8). Clontech에서는 CMV 유래 vector를사용하였을때클론간의발현차이를나타내거나발현정도가낮다고알려진 Jurkat cell을이용해 EF-1 alpha 버전을테스트하였다. EF-1 alpha promoter로 transactivator를발현시켰을때 83% 의 Jurkat 클론이높은유도발현을보였고, 2, 배이상의높은유도발현율을나타낸클론도 33% 나되었다 ( 그림 8). EF-1 alpha promoter는줄기세포의유전자발현에적합하며, Tet- On Inducible Expression (EF1 α version) 이 mouse embryonic stem cell에서효과적으로작용하는것을확인하였다 ( 그림 9). 2, 16, Jurkat Cells Dox + Dox 최적화된 Tetracycline Approved Serum system은감도가크게증가되었기때문에 tetracyclinefree가보장된 fetal bovine serum을선택하는것이매우중요하다. Clontech에서판매되는 serum은실제 Tet inducible cell line에서테스트를진행하여기저발현에영향을미치지않음이확인된제품이다. system에는 5 ml의프리미엄 Tet-Approved FBS가포함되어있다. G, it is essential to use fetal bovine serum that is func 그림 1. To ensure low background with, it is essential to use fetal bovine serum that is functionally tested. Clontech fers four such serum options, including serum that is sourced from US, Mexico, Australia, as well as US-sourced serum that is additionally tested for use in mouse embryonic stem cells. 12, RLU 8, 4, Clone no. 그림 8. (EF1α Version) provides inducible expression in hematopoietic cells. Jurkat cells were transfected with pef1α-tet using Xfect transfection reagent, stable clones were selected by limiting dilution. 18 stable clones were n tested for inducibility by transient transfection using P TRE -expressing luciferase. Six out eighteen clones showed more than 2, fold induction via transient transfection. References 1. Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89(12): Urlinger, S. et al. (2) Proc. Natl. Acad. Sci. USA 97(14): LӦw, R., Heinz, N., Hampf, M., Bujard, H. & Gossen, M. (21) BMC Biotechnology 1: Zhou, X., Vink, M., Klaver, B., Berkhout, B. & Das, A. T. Optimization Tet- On system for regulated gene expression through viral evolution. (26) Gene Ther. 13(19): A C Mouse Embryonic Stem Cells Dox No Dox B D 그림 9. (EF1α Version) provides inducible expression in stem cells. Mouse embryonic stem cells (ES-E14TG2a mes cells) were cotransfected with P TRE - ZsGreen1 pef1α-tet using Xfect Stem transfection reagent. The stem cells show ZsGreen1 expression only in presence Dox (Panel C). 제품명 Size Code Inducible Expression each Inducible Expression (EF1α Version) each Inducible Expression (Bicistronic Version) each Inducible Expression (with mcherry) each Inducible Expression (with ZsGreen1) each Tet Approved FBS 5 ml Doxycycline 5 g TetR Monoclonal Antibody (Clone 9G9) 2 μg License Notice [K45] 46 Life Science & Biotechnology 48

49 Mammalian Inducible Expression 가장빠르고간편한 Tet inducible expression system Tet-Express Inducible Expression o expression took place in absence Tet-Express treatment (Figure 1). 신속한셋업 /Tet-Off cell line을구축할필요없음 신속한발현유도 2시간만에최대발현의 8% 도달 Doxycycline-free 프로토콜 세포에 Tet-Express transactivator를직접첨가 RLU 16, 12, 8, Tet-Express No Tet-Express Tet-Express 유도발현시스템은기존 Clontech의강력한 Tet- On/Tet-Off시스템 (1 3) 보다더빠르고간편화된시스템으로 Tet- On/Tet-Off와달리, 단일 vector와 Tet-Express 단백질그리고 doxycycline-free 프로토콜로구성되어있다. 타겟유전자는 TRE 프로모터로조절되며세포배양배지에 Tet-Express를몇μl를첨가하면발현을유도할수있다. 따라서 Tet-Express시스템을적용하면테트라사이클린tetracycline으로전사를조절하는유도발현시스템을다양한세포에신속하게구축할수있으며, 특히클론선별과정을적용할수없는세포에도적용할수있다. Tet-Express란무엇인가? Tet-Express는 Clontech의 Tet-Off Advanced transcriptional activator (transactivator) 단백질로 self-transduction하도록변형되고최적화되었다. Self-transduction이란단백질번역경로를통해스스로세포막으로부터핵으로이동하는능력을의미한다. 테트라사이클린tetracycline 부재시 Tet-Express가프로모터에결합하여발현을활성화시킨다. 따라서유전자전사를활성화시키는데별도의독시사이클린doxycycline의투여가필요하지않다. Tet-Express와함께제공되는 Intensifier Reagent는단백질도입효율을높인다. 그림 1. Tet-Express rapidly induces gene expression from TRE promoters. HeLa cells containing a stably integrated luciferase expression cassette under control a TRE promoter were treated with Tet-Express compared to untreated cells from same HeLa cell culture at different time points. 88% maximal expression was achieved in just 2 hr. 광범위한세포에서높은발현유도와낮은백그라운드 Tet-Express 시스템은 transactivator를발현하도록별도의세포주를구축할필요가없기때문에복수의클론선별과정이필요없다. 따라서순차적인클론선별과정을거치기어려운 primary cell과같은세포주에특히더유용하다. Tet-Express는광범위한세포주에매우효과적이다 ( 그림 2). 1,4, 1,2, 1,, 4, 8, 5 min 15 min 3 min 1 hr 2 hr O/N 293T 1,2, 1,, 8, HeLa MCF7 HepG2 25, 35, 2, 15, 3, 25, 2, Tet-Express No Tet-Express RLU 6, Tet-Express Induction AcGFP1 6, 1, 15, 4, 4, 1, Tet-Express Protein P TRE Promoter Transcription 2, , 5, 5, Uninduced Tet-Express 시스템은 시스템과같이강력하게조절되는 P TRE 프로모터로타겟유전자를발현시킨다. 그러나 시스템과달리, self-transducing Tet- Express transactivator를세포에직접첨가하면되기발현이유도때문에 tetracycline transactivator를발현시키는 double-stable cell line을구축할필요가없다. 그림 2. Tet-Express works well with a broad range cell types. Cultures 293T cells, HeLa cells, MCF7 cells, HepG2 cells were transfected with a plasmid that expresses luciferase under control a TRE promoter. The next day, cells from four transfected cell cultures were treated with Tet-Express according to stard protocol incubated overnight in parallel with untreated cells from each transfected culture. On following day, treated untreated cells were assayed for luciferase activity. TRE 를포함하는프로모터중가장낮은기저발현 신속한유전자발현유도 Tet-Express 처리했을때와하지않았을때의유전자발현을 TRE 프로모터로조절되는 HeLa 세포내 luciferase 유전자발현을통해비교했다. Tet-Express 첨가시 2시간후 luciferase 발현이최대발현의 8% 이상으로신속하게증가하는것이확인되었으며 Tet-Express를처리하지않았을때는발현이나타나지않았다 ( 그림1). ptre vector는모든 와 Tet-Express 시스템에포함되어있다. 이 vector의 P TRE 프로모터는극도로낮은기저발현와발현유도시높은최대발현을유도한다 (4; 참고 teton3g). Minimal CMV 프로모터상류에위치한 19 bp tet operator 서열이 7회반복되어있으며, 이것을 tetracycline response element (TRE) 라고한다. tet operator repeats 서열은모든 Tet 시스템에서동일하지만 P TRE 의 junction sequences가고른간격이되도록변경되 47

50 Mammalian Inducible Expression 었고중앙부분이임의배치되어있다. 추가적으로, minimal CMV 프로모터의 element( 요소 ) 도일부변경되었다 ( 그림 3). 7 identical teto repeats (transactivator binding sites) Equal spacing romized central sequence 그림 3. The P TRE promoter is designed to ensure lowest basal expression any TRE-containing promoter. This promoter consists 7 identical teto repeats, separated by romized central sequences equal length. Minimal CMV promoter mutated to consensus P TRE 프로모터에내인성 endogenous 포유류전사요소가결합할수있는 사이트가부족하기때문에 transactivator 단백질이없을때는 minimal CMV 프로모터가비활성화된다. 따라서 TRE를포함하는프로모터중에서 P TRE 의기저발현이가장낮다. 기저발현은 western blot 으로검출되지않았고 transactivator가첨가되었을때최고수준으로발현이유도되었다 ( 그림 4). Tet-Express No Tet-Express 그림 4. Basal expression from PTRE promoter is undetectable by Western analysis. HeLa cells containing a Luciferase stably integrated tetracycline-inducible luciferase expression cassette were treated with Tet-Express according to stard protocol incubated overnight in b-actin parallel with untreated cells from same HeLa cell culture. The next day, lysates treated untreated cells were prepared blotted with antiluciferase anti-actin antibodies. promoter is undetectable by Western analysis. HeLa cells co 4 종류의 Tet-Express 시스템 Tet-Express 유도발현시스템 (P TRE vector 포함 ) 은또한 3종류의다른 vector 형태로도판매되고있다. 이 vector들은 Tet-Express Inducible Expression (Bicistronic Version), Tet-Express Inducible Expression (with mcherry) 과 Tet-Express Inducible Expression (with ZsGreen1) 형태로판매되고있다. 시스템구성품 Tet-Express (25 rxns Tet-Express Transactivator와 Intensifier Reagent 포함 ) P TRE 프로모터로목적유전자발현조절 vector 컨트롤 vector : P TRE 프로모터로 luciferase 발현조절 2종의선형화된선별마커 (hygromycin, puromycin) Tet Approved FBS Xfect transfection reagent References 1. Gossen, M. & Bujard, H. (1992) Proc. Natl. Acad. Sci. USA 89(12): Gossen, M. et al. (1995) Science 268(5218): Urlinger, S. et al.(2) Proc. Natl. Acad. Sci. USA 97(14): LӦw, R., Heinz, N., Hampf, M., Bujard, H. & Gossen, M. (21) BMC Biotechnology 1:81. 제품명 Code Vector Map SIZE Applications Tet-Express Inducible Expression ptre P TRE Tight MCS poly A each 유도발현이강력하게조절된다. Tet-Express Inducible Expression (Bicistronic Version) ptre-ires P TRE (B) MCS IRES MCS poly A each 2 개의유전자를유도발현시킨다. Tet-Express Inducible Expression (mcherry) ptre-mcherry each P Tight mcherry IRES poly A P TRE MCS 적색의형광단백질은 mcherry를목적유전자와함께발현시킨다. Tet-Express Inducible Expression (ZsGreen1) ptre-zsgreen1 녹색형광단백질인 ZsGreen1을 P Tight ZsGreen1 IRES poly A each 목적유전자와함께발현시킨다. P TRE MCS Tet-Express rxns rxns 48 Life Science & Biotechnology 48

51 Transfect a large amount active protein Virtually no cytotoxicity, unlike lipection Very high efficiency, even in stem or hematopoietic cells Simple protocol assay for your protein in just 2 hours Xfect Protein Transfection Reagent Our new Xfect Protein Transfection Reagent uses a cell-penetrating peptide developed at Clontech to bind transport active proteins directly into a wide variety mammalian cell types, including hard-to-transfect human suspension cell lines mouse embryonic stem cells. Rapid, high-efficiency, low-toxicity protein transfection Transfect a largeprotein amount Transfection active protein Reagent how does it work? What is Xfect Virtually cytotoxicity, unlikeis lipection Xfect Proteinno Transfection Reagent a modified peptide with cell-penetrating activity whose amino acid composition enables it to interact Very with high aefficiency, even stem orthis hematopoietic protein cargo intransport protein acrosscells a cell membrane barrier. Just incubate your protein interest with Xfect supplied buffer for 3 min, n apply inmixture to your cells 2 hours later, you are ready to assay for protein activity. in Simple protocol assay for your protein just 2 hours Xfect Protein Tranfection Reagent (cell-penetrating peptide) Your protein Extracellular Xfect Protein Transfection Reagent continued What is transfection efficiency Xfect Protein Transfection Reagent? Cytoplasm Th e transfection efficiency Xfect Protein Transfection Reagent can vary depending on cell type protein delivered. However, in all cell lines tested, Xfect outperforms Product C. When we transfected AcGFP1 (green fluorescent protein) into several different cell types using Xfect measured transfection efficiency using flow cytometry, more than 9% cells were transfected in 4 out 8 cell types that we tested 7 se 8 showed more than 8% transfection efficiency (Figure 6, Panel A). When immunluorescence microscopy was used to compare efficiency AcGFP1 transfection into HeLa cells using Xfect Product C (a leading competitor), a high level transfection was observed only for cells treated with Xfect (Figure 6, Panel B). Simple, rapid protein transfection with Xfect Protein Transfection Reagent. Xfect s cell-penetrating activity enables proteins to be transported across membranes mammalian cells. B A Xfect % positive Product C % positive HeLa Only Xfect Product C 12 1 % Positive cells Mammalian Expression s Rapid, high-efficiency, low-toxicity protein transfection Ordering Information 8 Product 6 Size Cat. No. Xfect 4 Protein Transfection Reagent 3 rxns 1 rxns NIH3T3 HEK293 HT18 HeLa CHO-K1 HL-6 Jurkat Clontech Laboratories, Inc. E14TG2A mes A Takara Company FigureBio 6. Protein transfection efficiencies across different cell lines: Xfect Protein Transfection Reagent vs. leading competitor, Product C. Panel A. Xfect Protein Transfection Reagent yields higher transfection effilines: ciencies than Product C across areagent broad range leading mammalian cells, including number rodent 그림 1. Protein transfection efficiencies across different cell Xfect Protein Transfection vs. competitor, Product C.a Panel A. Xfect Protein United States/Canada: Asia Pacific: Europe: +33.() Japan: +81.() For Research Only. Not cell for uselines, in diagnostic or rapeutic procedures.human Not for resale. Clontech, Clontech logo, allmouse or trademarks are property Use human hard-to-transfect suspension cells, embryonic stem cells. The cells were transfected with 4 μg recombinant Clontech unless noted orwise. 21 Clontech Laboratories, Inc. protein (eir racgfp1 or rdsred-express) using Xfect Protein Transfection Reagent or Product C according to manufacturers recommended Notice fl touorescent Purchaser The useprotocol. se products technologies may governed by one or more licensingby agreements. view specific current licensing information for any ClontechPanel product, please consult transfected Transfection effibeciency was assessed flow To cytometry one hour post-transfection. B. HeLa with 5 μg racgfp1 using Product C product s webpage on our website at Transfection Reagent yields higher transfection efficiencies than Product C across a broad range mammalian cells, including a number rodent human cell lines, hard-to-transfect human suspension cells, mouse embryonic stem cells. The cells were transfected with 4 μg recombinant fluorescent protein (eir or Xfect Protein Transfection using Reagent. racgfp1 rdsred-express) Xfect Protein Transfection Reagent or Product C according to manufacturers recommended protocol. Transfection efficiency ANY3741 IN or was assessed by flow cytometry one hour post-transfection. Panel B. HeLa transfected with 5 μg racgfp1 using Product C or Xfect Protein Transfection Reagent. Will Xfect Protein Transfection Reagent work for all cell types, including cell types that Code 제품명 Size cannot be easily transfected with? rxns Target cell choices arexfect not limited. We have successfully Protein Transfection Reagent transfected a variety mammalian cell types (human rodent) with high rxns which were transfected with close to 1% efeffi ciency, including hard-to-transfect human suspension cell lines (Jurkat,1 HL-6), ficiency (Figure 7). Mouse ES cells (E14TG2A) are transfected at an amazing 87% efficiency when using Xfect Protein Transfection Reagent (Figure 6, Panel A), despite fact that y cannot be transfected using Product C from a leading competitor. Xfect 12 Product C

52 Visually Monitor Cell Cycle Progression in Living Cells in Real-Time Single cells or complex tissues no fixation required! Easily precisely track cell-cycle progression in live cells Monitor phase transitions in cultured cells or live tissue Visualize cell shape during phase transitions Label whole cells or just nuclei Monitor stably expressed phase reporters over long time periods AmCyan1-hGeminin (1-11) SV4 polya signal P Tight-BI A B C P TRE mincmv-2 P mincmv-1 ptre-cellcycle 4826 bp mcherry-hcdt (3-12) SV4 polya signal G2 S M G1 Amp r puc ori 그림 1. Tightly controlled, simultaneous expression two Fucci probes allows complete visual tracking cell cycle. ptre-cellcycle is a bidirectional, tetracycline (Tet)-inducible expression vector that lets you inducibly express Fucci probes during all phases cell cycle (Panel A). In cells transfected with vector, Cdt1 fusion is visible through G1 phase, while Geminin fusion is visible from S through M phases (Panel B). A fl uorescence micrograph HEK 293 Advanced cells transfected with ptre-cellcycle vector cultured in presence Dox is shown in Panel C. pretrox-sg2m-cyan AmCyan-hGeminin (1-11) P P CMV IE Neo r PGK G2 M S G1 1 G1 S/G2 2 M 3 Early G1 mcherry-hcdt1(3-12) pretrox-g1-red P CMV IE P PGK Puro r G2 M S G1 mcherry-hgeminin(1-6) pretrox-sg2mcyto-red P CMV IE P PGK Puro r G2 M S G1 mcherry-hgeminin(1-11) 1 11 G1 S 12 pretrox-sg2m-red P CMV IE P PGK Puro r G2 M S G1 그림 2. Retroviral delivery is available for a variety Fucci probes. 그림 3. Cell cycle progression as visualized with Fucci probes. Clontech Fucci Probes Vector Phase: Color Localization Delivery Extra Benefits Cat. No. pretrox-sg2m-cyan S/G2/M: Cyan Nuclei Retroviral pretrox-g1-red G1: Red Nuclei Retroviral pretrox-sg2mcyto-red S/G2/M: Red Nuclei & Cytoplasm Retroviral Visualize cell shape pretrox-sg2m-red S/G2/M: Red Nuclei Retroviral ptre-cellcycle G1: Red S/G2/M: Cyan Nuclei Plasmid Tet-inducible; 2 Fucci probes in 1 vector

53 Accurate Cell Capture with CherryPicker s Don t just monitor cells expressing your protein or having your active promoter CAPTURE m, too! Don t just monitor cells expressing your protein or having your active promoter CAPTURE m, too! No easy access to a FACS sorter? Working with IRES vectors? Doing promoter studies? Want to capture cells expressing your protein interest? Doing gene expression studies? Want to capture cells with an active promoter? Select for gene expression or promoter activation in < 2 hr CherryPicker s use a membrane-targeted red fluorescent protein (CherryPicker) to identify your cells interest. These cells can be captured using our specially-formulated magnetic beads regrown into a more homogeneous population. Capture takes less than two hours, without time or expense a FACS sorter or or more elaborate cell sorting protocols. Code Products Size CherryPicker Assay Kit 6 Rxns CherryPicker Assay Kit 12 Rxns CherryPicker Cell Capture 6 Rxns CherryPicker Cell Capture (IRES) 6 Rxns Lenti-X CherryPicker Cell Capture 6 Rxns Lenti-X CherryPicker Cell Capture (IRES) 6 Rxns

54 Enzyme Immunoassay 골전환율 bone turnover: 골형성과골흡수의생화학마커 인간골격을이루는 26개의뼈는근육과다른신체기관을부착하거나지지하는물리적인골조역할을한다. 살아있는뼈는 65 % 의미네랄 ( 주로칼슘수산화인회석calcium hydroxyapatite 형태의인산칼슘calcium phosphate) 과 2% 의유기물 ( 대부분콜라겐타입 I ), 그리고 15 % 의물로구성되어있다. 그조직과구조는사람이만든건축물보다뛰어나며, 인간의경우평균 8년정도기능을수행한다골다공증Osteoporosis은 5세이상연령층의 12.5% 에서발병하는퇴행성뼈질환으로, 이연령대의 5% 이상이골다공증의위협을받을수있다. 골다공증은낮은뼈밀도와뼈조직의구조적인퇴화에의해특성을나타내며, 잦은골절상및추가합병증을래한다. 이연령대의여성들은남성들보다골다공증과연관된골절상이 4배정도더많이발생된다. 골다공증의연구와진단을위해과학자및의사들은골아세포 osteoblast의기능과골형성bone formation이나골흡수bone resorption를평가하는특정골대사마커 ( 활성골아세포를직접또는간접적측정하는제품 ) 의측정에의존하고있다. 다양한골대사마커들은성장여러단계에서나타나기때문에, 골아세포기능과골형성의다양한양상을모니터링하는데유용하다. TAKARA는혈청이나혈장에서두가지주요골형성마커 ( 오스테오칼신osteocalcin 및 procollagen type I C-peptides [PIP]) 를정량측정할수있는 5가지 EIA(enzyme immunoassay) 키트를제공하고있다. 골전환율bone turnover 연구에사용할다른 TAKARA 제품으로는뼈와연관된세포에서 ALP(Alkaline Phosphatase) [ 골형성 ] 와 TRACP(Tartrate-Resistant Acid Phosphatase) [ 골흡수 ] 의활성측정용 TRACP & ALP Double Stain Kit (Code MK3) 가있으며 ACP (Acid Phosphatase) 와 ALP 정량측정용 TRACP & ALP Assay Kit(Code MK31) 뿐만아니라오스테오칼신osteocalcin과 PIP에대한단일클론항체와다클론항체등이있다. 골형성bone formation 마커 ALP는유골osteoid 형성과무기질화mineralization에중요한역할을하는효소이다. 뼈특이적 ALP는골형성osteogenesis을촉진하는기능을가진오스테오칼신막당단백질membrane glycoproteins이며, 골아세포osteoblast 활성도의마커로잘알려져있다. 특히 ALP는피로인산염pyrophosphate ( 석회화calcification 위치에서결정화억제제 ) 뿐만아니라유기인산에스테르organic phosphate esters ( 무기인산염농도증가 ) 를분해한다. 혈청에서전체 ALP는다양한조직 ( 간, 뼈, 장, 비장, 신장, 태반 ) 에서유래한여러 dimeric isorm으로구성되어있다. 그러나건강한성인에서 는혈청 ALP의 5% 는간에서, 나머지 5% 는뼈로부터얻어진다. TAKARA의 TRACP & ALP Double-Stain Kit는 ALP와 TRACP 활성에따라골세포를각각다른색으로염색한다. 본제품은골세포샘플에서이두가지효소활성을시각적으로구분할수있도록하고, TRACP & ALP Assay Kit는 TRACP와 ALP의정량분석에사용된다. 골아세포osteoblast의두번째특이적마커는오스테오칼신osteocalcin (OC) 이며, 비콜라겐뼈기질단백질non-collagenous bone matrix proteins의약 15% 를구성하고있다. α-carboxylglutamic acid protein으로알려진 OC는작고 (5.9 kda), 비타민 K-의존적인 hydroxyapatite (Ca 2+ )- binding protein으로, 골아세포와치아의상아아세포odontoblasts ( 이세포는치아의상아질이된다 ) 에서만발현된다. OC의조직특이적발현은골형성에대한전반적인세포활성의훌륭한지표가된다. 이단백질의 17, 21 및 24에위치한 3 α -carboxyglutamic acid (Gla) 잔기들은칼슘결합을담당한다. 뿐만아니라 OC 합성은비타민 D와비타민 K 양쪽에의존적이며, 비타민 K가 glutamic acid 잔기들의 α-carboxylation을자극하는동안비타민 D는직접적으로 OC 합성을유도한다. 골형성에서 OC의정확한기능은잘알려져있지않지만, 피드백메커니즘을통해골리모델링에관여하는것으로추정된다. 혈청 OC 레벨은골형성속도와연관이있다. 그러므로 carboxylated OC (Gla-OC) vs. decarboxylated OC (Glu-OC) 의 immunoassay 측정은임상평가연구에유용하다. TAKARA에서는혈장내의 Gla/Glu OC 레벨, 세포 / 조직추출물또는세포배양상등액측정용 immunoassay kit 3종류를제공하고있다 (Code MK111, MK118 [for Human], MK126, MK146 [for Rat]) Procollagen Type I propeptide (N- 및 C-) 는골형성마커의세번째타입이다. 이러한프로펩타이드propeptide들은콜라겐 I 전구분자precursor molecules에서발견되는 NH-말단펩타이드서열이다. 이러한서열들은소포체endoplasmic reticulum 내에서프로펩타이드분자를삼중나선형태로감아돌리는것을촉진하는기능을한다. 콜라겐이분비되는동안에콜라겐삼중나선분자로부터프로펩타이드가쪼개지며, 이후삼중나선콜라겐들은세포밖콜라겐섬유로중합된다. 따라서자유프로펩타이드free propeptide의양은콜라겐분자합성의양을반영한다. 특히 procollagen type I carboxy-terminal peptide (PIP) 는골질환, 알코올성간질환, 간경변liver cirrhosis 및위의경성선암scirrhous (Borrmann type IV) adenocarcinoma 을포함한질환과콜라겐레벨의연관성을연구하기위한참고자료로유용하다. TAKARA는 2개의마우스단일클론 PIP 항체를이용하여샌드위치방 52 Life Science & Biotechnology 48

55 Enzyme Immunoassay 법의 EIA 키트인 Procollagen Type I C-Peptide EIA Kit (Code MK11) 제 공하고있으며, 혈장, 혈청, 세포배양추출물, 세포배양상등액및기타생체액biological fluid에서인간, 소또는개의 PIP 정량측정이가능하다. 골흡수 bone resorption 마커 뼈콜라겐의파괴에서파생된분해산물들은일반적으로골흡수bone resorption에대한마커로인식되고있다. 이러한분해산물들은히드록시프롤린hydroxyproline, PYD(pyridinoline) 그리고 DPD(deoxypyridinoline) 를포함하고있으며, 이러한물질뿐만아니라 BSP(bone sialoprotein) 와 TRACP와같은비콜라겐단백질또한이러한마커로이용되고있다. 골아세포osteoblasts와상아아세포odontoblasts에의해만들어진 BSP는뼈기질의비콜라겐부분의 5~1% 를구성하고있으며세포기질접착에중요한역할을하고, BSP 혈청레벨은골흡수과정과연관되어있는것으로추정된다. 두번째비콜라겐단백질 TRACP는파골세포osteoclast와모발상세포백혈병Leukemia hairy cells에특이적으로연관되어있다. 이효소는 5a 및 5b 2개의 sub-isorm으로존재하며, 파골세포osteoclast에는 TRACP-5b만특이적으로존재한다. TAKARA의 TRACP & ALP Double-Stain Kit 와 TRACP & ALP Assay Kit는뼈와관련된세포에서 TRACP와 ALP 활성을측정할수있다. 골대사는골형성및골흡수가상호균형적으로존재하기때문에이러한두개의마커를동시비교하는것은매우유용한방법이다. 요약골전환율bone turnover의생화학마커는인간과동물의골대사연구에유용하다는것이입증되었으며, 대부분의마커는뼈이외의조직에서발견되었다. 따라서, 생물학적체액내의마커는골형성 / 골흡수이외의신체프로세스에의해영향을받을수있다. 그럼에도불구하고 ELISA(enzyme-linked immunosorbant assays) 는혈청, 소변과생물체액을이용해골형성및골흡수마커를직접적이고도민감하게측정할수있다. TAKARA에서는 EIA, 염색키트그리고특정항체를포함한골전환율연구를위한다양한제품을제공하고있다. References Delmas et al. (2) The use biochemical markers bone turnover in osteoporosis. Osteoporos Int. Suppl. 6:S2-17. Yang, Y.J. Damron, T. (22) Histology Bone. emedicine ( Goddard, D. Kleerekoper, M. (1998) Osteoporosis symposium: Biochemical markers bone turnover. Postgraduate Medicine 14(4): TAKARA Product References Procollagen Type I C-Peptide EIA Kit (Code MK11) 1. Böhm, H., et al. (24) Collagen metabolism is a novel target neuropeptidemelanocytestimulating hormone. J. Biol. Chem. 279(8): Bourgier, C., et al. (25) Inhibition Rho kinase modulates radiation induced fibrogenic phenotype in intestinal smooth muscle cells through alteration cytoskeleton connective tissue growth factor expression. Gut 54: Hu, B., et al. (23) A nuclear target for interleukin-1 α : Interaction with growth suppressor necdin modulates proliferation collagen expression. PNAS 1(17): Kawaguchi, K., et al. (1999) Endogenous IL-1 α systemic sclerosis fibroblasts induces IL-6 PDGF-A. J. Clin. Invest. 13: Liu, X., et al. (24) camp-elevating agents adenylyl cyclase overexpression promote an antifibrotic phenotype in pulmonary fibroblasts. Am. J. Physiol. Cell Physiol. 286: C189-C Mori, K., et al. (1999) Dexamethasone enhances in vitro vascular calification by promoting osteoblastic differentiation vascular smooth muscle cells. Arterioscler. Thromb. Vasc. Biol. 19(19): Undercarboxylated Osteocalcin (Glu-OC) EIA Kit (Code MK118) 1. Lee, A., et al. (2) Measurement osteocalcin. Ann. Clin. Biochem. 37: Schurgers, L.J., et al.(24) Effect vitamin K intake on stability oral anticoagulant treatment: dose-response relationships in healthy subjects. Blood. 14(9): Schurgers, L.J., et al. (22) Novel effects diets enriched with corn oil or with an olive oil/ sunflower oil mixture on vitamin K metabolism vitamin K-dependent proteins in young men. J. Lipid Res. 43: Takahashi, M., et al. (21) Effect vitamin K /or D on undercarboxylated intact steocalcin in osteoporotic patients with vertebral or hip fractures. Clin. Endocrin. 54(2): 219. Gla-Type Osteocalcin EIA Kit (Code MK111) 1.Barroga, E., et al. (1999) Induction osteosarcoma functional differentiation growth inhibition in vitro canine by 22-oxacalcitriol, calcitriol all-trans retinoic acid. J. Vet. Med., Ser. A. 46 (9): Koyama, N., et al. (1991) A one step swich enzyme immunoassay for gammacarboxylated osteocalcin using monoclonal antibodies. J. Immunol. Methods. 139(1): Orui, H., et al. (21) Effects bone morphogenetic protein-2 on human tumor cell growth differentiation: a preliminary report. J. Orthopaedic Sci. 5(6): Schurgers, L.J., et al. (24) Effect vitamin K intake on stability oral anticoagulant treatment: dose-response relationships in healthy subjects. Blood 14(9): Tsukamoto, Y., et al. (2) Prolonged intake dietary fermented soybeans (natto) with reinforced Vitamin K2 (menaquinone-7) enhances circulating α -carboxylated osteocalcin concentration in normal individuals. J. Health Sci. 46(4): Yamaguchi, M., et al. (21) Prolonged intake islavone- saponin-containing soybean extract (nijiru) supplement enhances circulating-carboxylated osteocalcin concentrations in healthy individuals. J. Health Sci. 47(6):

56 Enzyme Immunoassay Crocodile miniworkstation 최강의효율과성능, 컴팩트한전자동 ELISA 분석기기 일반적으로 ELISA 실험은 1시간이상이소요되기때문에실험을진행하는동안다른중요한실험과병행하기어렵다. Crocodile은 reading 기능만을갖고있는 ELISA reader와같이컴팩트한사이즈이지만 5종의장비 (dispenser, shaker, incubator, washer, 및 reader) 가하는기능을모두수행할수있다. 따라서분주에서부터데이터수집 (reading) 까지 plate를옮길필요없이자동으로진행할수있기때문에시간을절약할수있다 간편한가동준비세척병, 폐액병그리고시약병이기기오른쪽에장착되어있어교체하기쉽고, 몇분내로기기를가동시킬수있다. 쉬운기기관리와유지직관적인디자인으로시스템의세척이나튜브의교체를쉽게할수있다. 또한보증기간이후서비스계약을체결하면기기관리서비스를받을수있다. 간편한장비설치모든장비의모듈이하나의박스에제공된다. 전원이나컴퓨터연결도간편하게설치할수있으며, 세척이나폐액처리를위한튜브가미리설치되어있기때문에손쉽게세척병과폐액병을설치할수있다 Dispenser 2. Shaker 3. Incubator 4. Washer 5. Reader 철저한검사 Crocodile은설치검사Installation Qualification, 작동검사Operation Qualification 그리고성능검사Performance Qualification의 3가지검사항목을통과한후출하된다. 기기특성에따라세분화된점검리스트로그결과가보고된다. 컴팩트한사이즈 27cm 공간만있으면설치할수있을만큼 Crocodile은컴팩트하다. 만약기기에시약병을설치하려한다면일반적인 ELISA reader 의규격인 4cm 정도면충분하다. 효율적인무인자동화시스템 Crocodile은무인가동시스템으로설계되어 sample microplate를기기에삽입하고프로토콜을선택하여가동시킨후에는모든실험이전자동으로진행되기때문에별도의작업이필요하지않다. 품질관리규정준수 Crocodile은 DIN ISO 13485(Quality Management for Medical Devices) 를준수하며개발되었다. 모든과정이철저한리스크분석을통해진행되었으며원자재선택부터조립과정까지철저한감독하에이루어진다. 최종조립된기기는다양한테스트를거치게되고이를통과한후성능이명시된기기분석증명서 (certificate analysis) 가첨부된다. 다양한 ELISA 프로토콜적용가능 Crocodile은사용자를고려한소프트웨어로다양한 ELISA 프로토콜을적용하기쉽다. 특히다섯가지장비의기능을갖고있기때문에분주단계를변형하거나세정과정을여러번반복하도록다양하게프로토콜을선택할수있다. 각단계를설정하는게쉬울뿐만아니라선택한실험과정은스크린에서그래픽으로전체과정을확인할수있다. 높은호환성 Crocodile은다양한 ELISA 방법 (swich법, competitive법등 ) 에적합하며시약병홀더에는다양한모양과크기의시약병도장착가능하다. The 8-well parallel washer provides consistent washing performance 4 high-precision reagent dispensers 54 Life Science & Biotechnology 48

57 Enzyme Immunoassay Crocodile Technical Data General specifications Sample format Dimensions (WxDxH) Weight Interface Power Requirements Operating Temperature Humidity 1 x 96 well microplate (SBS stard) 26.3 x 62.6 x 25.8 cm; 1.4 x 24.6 x 1.2 inch 14.5 kg; 32 lbs USB interface 24 V 5 Hz, 11 V 6 Hz 1 3 C; 5 86 F 1 8% (non condensing) Incubator Temperature Range Temperature Stability Incubation Time Temperature Monitoring Ambient C ± 1 C across plate at 37 C Programmable Yes The assay reagents are kept firmly in place by flexible bottle holders. Buffer waste bottles consume little bench space Reader Dynamic Range Spectral Range Filter Slots 8 Reading Channels Precision Accuracy 3. OD 4 69 nm (pre-installed filters: 45, 45, 492, 62 nm) 8 independent photometric channels plus reference channel, Mono bichromatic reading 1.5 % CV (.1 to.5 OD) 1 % CV (.5 OD to 2. OD) 1.5 % CV (2. 3. OD) ±.1 OD or 2.5 % (whichever is greater) Dispenser Type Volume Range Precision Accuracy Reagent Support 4 independent precision pumps 2 1 μl 2 % at 1 μl 5 % at 1 μl Any bottle or vial type can be attached Washer Manifold Configuration Dispense Volume Wash Modes Wash Buffer Capacity Waste Liquid Container Autoclavable 8 way manifold with 2 parallel needles 5 1 μl Multiple settings: Sweep mode, soak only, prime only, top bottom wash 3 containers for different wash solutions Input connectors for users external bottles, any size Shaker Shaking Independent linear motion 14 2 Hz PC Requirements Required Hardware Windows computer with free USB port. Please inquire for supported Windows versions 기기사양은사전공지없이변경될수있습니다. 55