Lentivirus Systems & Tools Lentivirus Transduction Tools 렌티바이러스농축시약 Lenti-X Concentrator 짧고간편한프로토콜 Ultracentrifugation 불필요 최대 1 배농축가능, ~9% 회수율 단순한프로토콜

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1 Lentivirus Systems Lentivirus Systems & Tools Lentivirus Systems 최대 ~1 8 IFU/ ml의매우높은역가의렌티바이러스조제가능 48 시간이내에 VSV-G pseudotype의 lentivirus 획득 5 개의 vector로분리된 packaging mix를이용하여바이러스감염에대한안정성확보 Clontech의 Lenti-X Expression System (Code 63164) 은광범위한세포에높은역가로적용할수있으며발현효율을높일수있도록한단계더발전된렌티바이러스시스템이다. RNA 바이러스계열의재조합레트로바이러스중하나인렌티바이러스는비분열세포와줄기세포, 뇌, 간, 근육과같은 in vivo 조직등거의모든포유류세포를감염및형질전환하여목적단백질을안정적으로발현시킨다. 최적화된 Lenti-X Vector Lenti-X vector는렌티바이러스의생산과복제에필요한 LTRs과패키징서열을가지고있을뿐만아니라이식유전자transgene의발현과역가를향상시킬수있는요소를포함하고있다. WPRE 서열은 93T cell 에서의바이러스패키징을향상시켜역가를증가시키고성숙한 mrna 의생산을촉진함으로써목적세포에서의 cdna transgene의발현을높인다. cppt element 는바이러스게놈을핵안으로삽입시키는과정의효율을증가시켜형질전환효과를향상시킨다. Lenti-X system은항시발현하는 pcmv기반의발현 (Lenti-X Expression System) 이지만 Tet-On/Off 발현의유도발현시스템 (Lenti-X Tet-On/Tet-Off Expression System) 또는형광단백질과융합한재조합단백질발현시스템 (AcGFP1 또는 DsRed-Monomer) 과같이다양한발현에적용할수있다. 최고의성능을갖는패키징시스템시스템에최적화된구성성분들간의시너지효과에의해 Lenti-X HTX Packaging System은뛰어난바이러스생산능력을갖는다 ( 그림 1). 첫째, Lenti-X HTX Packaging Mix는렌티바이러스의패키징에필수적인구성유전자가최적의비율로포함되어있어바이러스를고효율로생산가능하다. 또한이구성요소들이각각의 vector로분리되어있어바이러스의자가복제를억제할수있으므로실험자의감염가능성을현저히줄일수있다. 둘째, Tet-off 전사촉진에의한 Tetracycline-responsive promoter elements(tres) 조절로렌티바이러스패키징의핵심구성요소가고발현되어높은역가의바이러스를획득가능하다. 셋째, Clontech의렌티바이러스시스템에최적화된나노입자기반의트랜스펙션시약인 Xfect는효과적으로유전자를도입시켜고효율의렌티바이러스를생산한다. Xfect를 Lenti-X 93T 세포에적용시, 최대 95% 이상의트랜스펙션효율을확인하였다. 렌티바이러스관련모든제품군보유 Lenti-X system은거의모든세포에적용할수있으며특히형질도입이어려운세포에유전자를도입하여 stable cell line이나유도발현시스템을구축할때매우유용하다. 추가로 Lenti-X 시스템에포함된 Lenti-X GoStix을이용하면 3 초 ~ 1 분이내에렌티바이러스의감염역가가충분한지, 아닌지확인할수있으며만약역가가충분하지않다면 Lenti-X Concentrator를이용하여농축할수있다. 그외 Clontech에서는렌티바이러스를정제하는 Lenti-X Maxi Purification Kit나역가를측정하기위한 Lenti-X qrt-pcr Titration Kit, provirus의역가를측정하는 Lenti-X Provirus Quantitation Kit과같은제품을통해효과적으로유전자도입이가능하게지원하고있다. Gene of Interest A 75 Clontech s Lenti-X P CMV IE 5' LTR Ψ Puro R WPRE P PGK RRE cppt MCS tta 3' LTR Counts 5 5 Untransduced Transduced Xfect transfection Lenti-X HTX Packaging Mix ¹ 1² FL1-H Competitor s packaging system Untransduced 1³ 1 4 Lenti-X 93T cells Counts 5 5 Transduced 1. Collect virus after 48 hr. Transduce host cells 그림 1. 4th generation lentiviral packaging system. A lentiviral vector and the Lenti-X HTX Packaging Mix are cotransfected into 93T cells. High titer lentiviral supernatants are ready for use 48 hr after transfection. 1 1¹ 1² FL1-H 1³ 1 4 Lenti-X ( ) and a packaging system from a competitor 그림. High infectivity of supernatants produced by Lenti-X. Lenti-X (Panel A) and a packaging system from a competitor (Panel ) were used to generate virus containing a vector system for expressing the ZsGreen1 fluorescent protein. 1 μl of supernatant from system was used to transduce La cells. ZsGreen1-positive cells were quantified by flow cytometry Lenti-X transduced the majority of cells, whereas the other system transduced a small percentage of the cells. Life Science & iotechnology 5

Lentivirus Systems & Tools Lentivirus Transduction Tools 렌티바이러스농축시약 Lenti-X Concentrator 짧고간편한프로토콜 Ultracentrifugation 불필요 최대 1 배농축가능, ~9% 회수율 단순한프로토콜 : mix, wait, spin Lenti-X Concentrator

Lenti-X Concentrator 는 Clontech 의 Lenti-X Systems 을포함한대부분의렌 티바이러스에적용가능하다. Ultracentrifugation 방법에비해간편함 Ultracentrifugation 를이용하여렌티바이러스를농축하는것보다 Lenti-X Concentrator 를이용하는것이보다빠르고편리하며효과적 이다 ( 표 1)

2 Lentivirus Systems & Tools Lentivirus Transduction Tools 렌티바이러스농축시약 Lenti-X Concentrator 짧고간편한프로토콜 Ultracentrifugation 불필요 최대 1 배농축가능, ~9% 회수율 단순한프로토콜 : mix, wait, spin Lenti-X Concentrator 를이용하면빠르고쉽고효과적으로렌티바이 러스농축이가능하다. 이프로토콜은렌티바이러스상층액에 Lenti-X Concentrator reagent 를첨가하고, 3 분 (~overnight) incubation 후에일반 centrifuge 를이용하면 ( 그림 3) ultracentrifugation 없이 1 시간이내에최대 1 배까지렌티바이러스농축이가능하다. Lenti-X Concentrator 는 Clontech 의 Lenti-X Systems 을포함한대부분의렌 티바이러스에적용가능하다. Ultracentrifugation 방법에비해간편함 Ultracentrifugation 를이용하여렌티바이러스를농축하는것보다 Lenti-X Concentrator 를이용하는것이보다빠르고편리하며효과적 이다 ( 표 1). 표 1. Lenti-X Concentrator vs. Ultracentrifugation Feature Lenti-X Concentrator Ultracentrifugation Easily Scalable Yes No Specialized Equipment No Yes Time Required ~1 hr 4 hr to overnight Ease-of-Use Yield >9% >9% 렌티바이러스상층액정제로오염물질제거 Lenti-X Maxi Purification Kit Add Lenti-X Concentrator to clarified viral supernatant. Gravity column 방식을이용하여렌티바이러스입자손상을방지 세포오염물질을제거하여 transduction 효율높임 최대 1 배의농축효과와 6 ~ 8% 의회수율 Incubate at 4 C for 3 min to overnight Centrifuge at 1,5 x g at 4 C for 45 min Collect pellet and resuspend in 1/1 to 1/1 original volume Store at 8 C Titrate Infect targets 그림 3. The Lenti-X Concentrator protocol. Add Lenti-X Concentrator reagent to clarified viral supernatant, incubate for 3 min to overnight at 4 C, and spin. That s it Lenti-X Maxi Purification Kit를이용하면 crude한렌티바이러스상층액을 gravity column-base 방법을이용하여빠르고간단하며효과적으로렌티바이러스를정제할수있다. Filter를이용한정제방법은렌티바이러스입자에손상을주거나정제량을감소시키는데비하여 gravity column 방식은실험과정이빠르고간편하며효과적이다. Gravity column은세척과정동안 column을통해부착되지않은물질들이통과되고상층액내에바이러스입자들은남아있게되어, 정제된바이러스는온전한형태와기능을잃지않게된다. 이키트를사용하여바이러스를정제하는과정은 plasmid DNA를정제하는것만큼간편하다. 3 ml 3 μl 1 9 Titer (IFU/ml) ind Wash Elute 1 6 Total IFU: efore After 3.8 x x 1 7 그림 5. The Lenti-X Maxi Purification Kit allows you to generate high yields of purified lentivirus from crude packaging cell supernatants. The gravity column-based method (bind, wash, elute) is extremely simpleand effective, and preserves virus infectivity much better than filter-based methods. 그림 4. Efficient concentration with minimal loss. Lentiviral supernatant from a plvx-zsgreen1 vector was concentrated from 3 ml down to 3 μl using the Lenti-X Concentrator reagent, which reflected a 1-fold increase in viral titer. Measuring the total amount of virus contained in sample indicated that the resuspended pellet captured 9% of the virus present in the original sample. Samples were titrated using HT18 cells and analyzed by flow cytometry 48 hr post-transduction. 렌티바이러스를정제해야하는이유렌티바이러스의정제는실험에영향을미칠수있는세포오염물질을제거할수있다. 정제되지않은렌티바이러스상층액은잔존 plasmid DNA, 알려지지않은형질도입저해제, 세포내단백질과혈청단백질, 고도의면역성바이러스단백질, 핵산, 바이러스단편등을포함하고있 1

Lentivirus Systems 다. 민감한목적세포또는 in vivo 적용을위해서는렌티바이러스를사용하기전에정제하여오염물질을제거하는과정이필요하다. 정제된바이러스를사용하면 pseudo-transduction 도방지할수있다. 간단한프로토콜 Lenti-X Maxi Column은사용하기편리하도록구성되어있다.

Filter system보다뛰어난회수력과순도바이러스정제의핵심은바이러스의감염력을유지하고뛰어난바이러스회수능력을발휘하는것이다. 일반적으로 Lenti-X column은시료로부터 6% 이상의감염력을지닌바이러스가정제된다 ( 그림 6).

Instantly Test for Lentivirus Lenti-X GoStix. 3 초 ~ 1 분이내에렌티바이러스의역가판단.

붉은색밴드가선명하게나타나는경우의역가는 ~5 x 1 5 IFU/ml* 이다. * 역가측정은 HT-18 cell 을렌티바이러스를형질도입후 flow cytometry 이용하여측정 하였다. * 실험의민감도는사용하는역가측정방법이나렌티바이러스패키징시스템에따라서달 라질수있다. 4.6 x 1 3 IFU/ml 4.

6 x 1 3 IFU/ml Purification Method Yield (% of Input) qrt-pcr Fluorescence (RNA copies) (IFU) RNA copies IFU Ease of Use Lenti-X Gravity column 84.7 61.6 **** Vendor S Syringe filter 15.1 18.

Cells, and a plvx vector expressing Clontech s ZsGreen1 fluorescent protein, a clear was generated Control by a diluted supernatant containing ~5 x 1 5 IFU/ml* (as measured by flow cytometry of

3 Lentivirus Systems 다. 민감한목적세포또는 in vivo 적용을위해서는렌티바이러스를사용하기전에정제하여오염물질을제거하는과정이필요하다. 정제된바이러스를사용하면 pseudo-transduction 도방지할수있다. 간단한프로토콜 Lenti-X Maxi Column은사용하기편리하도록구성되어있다. 상층액 (9 ~ 45 ml) 에 1 x binding buffer를첨가한후 column에넣으면중력에의해시료와 buffer가통과하고, 회 column 세척과정동안불순물들은제거후, 3 ml의 elution buffer 로정제된렌티바이러스를회수한다. Filter system보다뛰어난회수력과순도바이러스정제의핵심은바이러스의감염력을유지하고뛰어난바이러스회수능력을발휘하는것이다. 일반적으로 Lenti-X column은시료로부터 6% 이상의감염력을지닌바이러스가정제된다 ( 그림 6). Anion exchange-based membrane system은 Lenti-X column 보다낮은회수능력을보이며종종 % 이하의감염력을지닌바이러스가정제되는경우도있다. Lenti-X column은 filter system보다높은바이러스순도와매우적은단백질검출을보인다. Filter 정제방식은바이러스와함께정제된외부단백질을다량포함하고있다 ( 그림 7). Instantly Test for Lentivirus Lenti-X GoStix. 3 초 ~ 1 분이내에렌티바이러스의역가판단. 바이러스회수최적타이밍판단 Lenti-X GoStix 으로측정한민감도범위 Clontech 의 plvx vector 중 ZsGreen1 형광단백질을발현하는렌티바 이러스벡터와 Lenti-X HTX Packaging System, Lenti-X 93T Cells 을이용하여렌티바이러스를제작후 Lenti-X GoStix 으로바이러스의 역가를측정하였다. 붉은색밴드가선명하게나타나는경우의역가는 ~5 x 1 5 IFU/ml* 이다. * 역가측정은 HT-18 cell 을렌티바이러스를형질도입후 flow cytometry 이용하여측정 하였다. * 실험의민감도는사용하는역가측정방법이나렌티바이러스패키징시스템에따라서달 라질수있다. 4.6 x 1 3 IFU/ml 4.6 x 1 4 IFU/ml A yield (%) Crude Flow Wash Elution 4.6 x 1 3 IFU/ml Purification Method Yield (% of Input) qrt-pcr Fluorescence (RNA copies) (IFU) RNA copies IFU Ease of Use Lenti-X Gravity column **** Vendor S Syringe filter ** V i r a l T r a n s d u c T i o n Sensitivity in a range you care about When Lenti-X GoStix were used to test viral supernatants produced Test using the Lenti-X HT Packaging System, Lenti-X 93T Cells, and a plvx vector expressing Clontech s ZsGreen1 fluorescent protein, a clear was generated Control by a diluted supernatant containing ~5 x 1 5 IFU/ml* (as measured by flow cytometry of transduced HT-18 cells). * Test sensitivity may vary when compared to different titration methods or when using different lentiviral packaging systems. 4.6 x 1 4 IFU/ml 4.6 x 1 5 IFU/ml 4.6 x 1 6 IFU/ml 그림 6. Lenti-X Maxi Purification Kit yields are Test far higherthan the yields of filter-based methods. Panel A. contentin the indicated Lenti-X column fractions Control was tracked using either Lenti-X qrt-pcr (RNA copies) or flow cytometry/fluorescence (IFU) titration. The mean values from seven experiments are shown. Panel. In a headto-head comparison, Lenti-X column purification recovers more virus than a filter-based method.far fewer contaminating proteins at equivalent IFU than either sample prepared from the filter-based systems. Real Time PCR 방법을활용한역가측정 You may also be interested in Lenti-X Concentrator Lenti-X qrt-pcr Titration Kit kd M Lenti-X Vendor M Vendor S 그림 7. The Lenti-X Maxi Purification Kit yields highly purified lentivirus. Equivalent amounts of purified virus (1 x 1 5 IFU) prepared using either the Lenti-X Maxi Purification Kit (Lenti-X) or a filter-based system (Vendor M & Vendor S) were subjected to SDS-PAGE and silver-stained. The Lenti-X sample Incubate clearly at 4 Ccontained for 3 min far fewer contaminating proteins to overnight at equivalent IFU than either sample prepared from the filter-based systems. Increase titers by 1-fold Simple protocol: mix, wait & spin Scalable for supernatants of any volume or titer Add Lenti-X Concentrator to clarified viral supernatant. 4 시간만에렌티바이러스의역가 (RNA 역가 ) 측정가능. 정확한역가측정을통해재현성있는바이러스감염실험가능 Lenti-X qrt-pcr Titration Kit 을이용하면 RNA 를정제후, SYR Green I 법을이용한 One-Step qrt-pcr 로 4 시간만에렌티바이러스의 Store at 8 C 역가를측정할수있다. 표준곡선작성에필요한 control RNA는제품에 포함되어있다. 또한 FACS 나콜로니선별등을통해바이러스의생물학 Centrifuge at 1,5 x g at 4 C for 45 min Collect pellet and resuspend 적역가 (IFU/ml) 와 RNA 역가 (copies/ml) 와의상관관계 (copies/ifu) 를 in 1/1 to 1/1 original volume Titrate Infect targets 미리계산해두면, 측정된 RNA 의역가를통해 시간만에생물학적역 Control 가를추정하여 MOI(Multiplicity of Infection) 를결정할수있다. Test Life Science & iotechnology 5

Lentivirus Systems & Tools ELISA 법을활용한역가측정 Lenti-X p4 Rapid Titer Kit. 간단하고빠른 ELISA 기반의역가측정방법 Lenti-X p4 Rapid Titer Kit 는 ELISA 방법을이용하여 HIV-1 기반 의렌티바이러스상층액에서역가를측정한다.

4 Lentivirus Systems & Tools ELISA 법을활용한역가측정 Lenti-X p4 Rapid Titer Kit. 간단하고빠른 ELISA 기반의역가측정방법 Lenti-X p4 Rapid Titer Kit 는 ELISA 방법을이용하여 HIV-1 기반 의렌티바이러스상층액에서역가를측정한다. 바이러스상층액에존재 하는 p4 capsid 단백질의잔존량을측정하며, 이때 p4 의양은바이러 스역가와직접적인상관관계가있다. 렌티바이러스상층액를 anti-p4 capture antibody 로코팅된 96-well plate(1 개의 8-well strip 으로분 리가능 ) 에넣고, 세척후결합된 p4 는 biotinylated anti-p4 차항 체와 streptavidin-hrp, 발색시약을첨가하여측정한다. p4 control 을이용하여표준곡선을만들고, p4 를동량으로조정하여상층액의역 가를산출한다. 러스카피수측정을통해바이러스의실제유효한역가 ( 예 : 효과적인역가 ) 를확인하고정확한 MOI(multiplicity of infection) 로타겟세포에감염이가능해진다. 예측가능하고재현성있는유전자발현렌티바이러스를이용하여타겟세포에목적유전자를도입할때, 바이러스의 MOI가증가하면유전자의발현레벨이증가한다. 이는보다많은렌티바이러스카피수가세포의 genomic DNA로삽입되기때문이다 ( 그림 9). 효과적인렌티바이러스 MOI는각각의 cell line에따라서달라질수있다 ( 그림 1). Lenti-X Provirus Quantitation Kit를이용하면유전자레벨, cell line의종류, 실험시기와상관없이형질도입효율을평가할수있다 6, p4 는무엇인가? 재조합렌티바이러스를연구목적으로제작할경우, 3 세대또는 4 세대 패키징방법을이용한다. 재조합렌티바이러스의구조단백질은 VSV-G 또는 ecotropic envelope 단백질, 매트릭스단백질과바이러스코어단 백질로구성되어있다 ( 그림 8). Gag 유전자는바이러스 capsid 단백질 (p4) 과 nucleocapsid 단백질 (p6 과 p7), 매트릭스단백질 (p17) 을코딩하 고있다. 이구조단백질중, Lenti-X p4 Rapid Titer Kit 는바이러스 상층액에포함되어있는 p4 단백질의양을측정한다. Envelope (VSV-G or Eco) Reverse transcriptase MFI 5, 4, 3,, 1, Provirus copy no. 그림 9. Higher proviral content is associated with higher expression levels. Provirus content in the genomic DNAs of HT18 cells transduced at either high or low MOI were determined using the Lenti-X Provirus Quantitation Kit. AcGFP1 expression levels (MFI) were determined using flow cytometry. The figure represents data pooled from two groups having either low-copynumber (<3; n = 8) or high-copy-number (>9; n = 7) proviruses. RNA genom Provirus copy no HT18 HEK 93 La Cell line Capsid/p4 Figure 1. p4 is located in the lentiviral capsid and is one 그림 8. p4 is located in the lentiviral capsid and is one of 4 proteins encoded by the HIV-1 gag gene. 그림 1. Equivalent MOI in different cell types can result in different provirus copy numbers. HT18, La, and HEK 93 cell cultures were transduced at equivalent MOIs using a single lentiviral stock (1 μl). The provirus copy numbers in the genomic DNAs of the transduced cell populations were determined using the Lenti-X Provirus Quantitation Kit. 삽입된렌티바이러스의카피수를측정 Lenti-X Provirus Quantitation Kit. Transduction 된세포로부터 integrated lentivirus(provirus) 의카피 수를측정. 실험조건이나세포종류가달라져도형질전환시의감염력을일정하 게유지하기위해사용 렌티바이러스를감염시킨세포의 genomic DNA 내에삽입된렌티바이 러스 (provirus) 의카피수를신속하게측정하기위한제품이다. 프로바이 Inverted PCR 분석으로 provirus 검출확인 Lenti-X Provirus Quantitation Kit로분석된 provirus의카피수의결과를확인하기위해 proviral insertion junctions 부분을 invert PCR(iPCR) 로증폭했다 ( 그림 11). 예상대로 low-copy-number 클론에서는단일 PCR 산물 (~15 bp) 이생성되었으며, mixed polyclonal populations에서는 1 ~ 5bp 사이의다양한 PCR 산물이 smears 하게검출되었다. 이결과는 proviral insertion이많이일어났음을의미한다. 두종류의실험결과가일치하였기때문에, Lenti-X Provirus Quantitation 방법이효용성이있음을확인하였다. 3

Lentivirus Systems bp Clone Popn. M 1 3 4 5 NTC M 5 ure 3. Proviruses amplified by inverted PCR correspo 그림 11. Proviruses amplified by inverted PCR correspond to qpcr copy number.

The DNA samples were also subjected to inverted PCR analysis to amplify individual provirus junction sequences using vector-specific primers (1). NTC = no template control.

Starter Kit 은 magnetic separator 를포함 Lenti-X Accelerator 는 magnetic bead 기술을기반으로렌티바이러스 와 MSCV 레트로바이러스를포함한 MMLV 레트로바이러스의형질도입 을촉진할수있도록제작되었다.

5 Lentivirus Systems bp Clone Popn. M NTC M 5 ure 3. Proviruses amplified by inverted PCR correspo 그림 11. Proviruses amplified by inverted PCR correspond to qpcr copy number. Provirus copy numbers were determined for genomic DNA purified from 16 stably transduced HT18 cells representing either individual clones (Lanes 1 3) or polyclonal populations (Lanes 4 & 5). The DNA samples were also subjected to inverted PCR analysis to amplify individual provirus junction sequences using vector-specific primers (1). NTC = no template control. Note: Not all proviruses can be amplified with this method. Polybrene 없이 5 분이내에 magnetic bead 를이용하여형질도입 Lenti-X Accelerator. 렌티바이러스나 MMLV 레트로바이러스의빠른 transduction 유도.Stem cell 과같이민감한세포에최적.Starter Kit 은 magnetic separator 를포함 Lenti-X Accelerator 는 magnetic bead 기술을기반으로렌티바이러스 와 MSCV 레트로바이러스를포함한 MMLV 레트로바이러스의형질도입 을촉진할수있도록제작되었다. Polybrene 을이용할경우, overnight 으로노출시켜야하기때문에시간이오래걸리고독성을나타내는반면, Lenti-X Accelerator 는세포를바이러스상층액 (viral supernatant) 에 5 분만노출시키면되기때문에 stem cell 과같은민감한세포적용에최 적이다. 빠르고효과적인형질도입 Lenti-X vector 를이용하여형질도입효율을비교해보면 Lenti-X Accelerator 는 polybrene 을이용한것과달리불과 5 분만에높은형질 도입효율을보인다 ( 그림 1). Magnetic Separator Lenti-X Accelerator Starter Kit 에는세포배양용플레이트에이용할 수있는 magnetic separator 가포함되어있다 ( 그림 13) Positive cells (%) Copy no Positive cells MFI Negative control Lentivirus + Polybrene 5 min Lentivirus + Lentivirus + Lenti-X Accelerator Polybrene O/N 그림 1. Lenti-X Accelerator provides high transduction efficiency in a 5 min protocol. Lentiviral transduction of HT18 cells was carried out for 5 min with Lenti-X Accelerator beads after a min incubation to bind the beads to the virus and for 5 min or overnight with Polybrene. After the cultures were grown for an additional 7 hr at 37 C, the number of transduced cells was determined by flow cytometry. 5 min,5, 1,5 1, 5 Mean fluorescent intensity (MFI) 형질도입방법을변화하라 Viral Receptor oosters 그림 13. A magnetic separator is included with the Starter Kit.. 높은형질도입효율 - 일시적으로목적 cell 의 viral receptor 농도를증가. 간단한프로토콜 타겟세포에효과적으로바이러스를도입하기어려울때가있다. 실험실 에서주로사용되는 3 종류의바이러스 ( 레트로바이러스, 렌티바이러, 아 데노바이러스 ) 는세포표면의수용체단백질을통해서세포내로도입된 다. 세포표면에수용체단백질이제한적으로존재하면형질도입효율이 낮아진다. Clontech 의 Viral Receptor ooster 기술은쉽고빠르게타 겟세포표면에수용체단백질을증가시켜바이러스의형질도입효율을 증가시킬수있다. Viral Receptor oosters 의역할 Viral receptor booster 는 exosome-like vesicles 또는 microvesicles 형 태로세포의세포막에 viral receptor protein 을일시적으로증가시킨다 ( 그림 14). 타겟세포표면에바이러스수용체단백질이증가함으로써형 질도입효율이증가된다. 1. Target cells lack receptor Target cell 3. Receptor oosters fuse with target cells to increase the concentration of receptor 그림 14. The principle of Viral Receptor ooster technology. Viral Receptor oosters are concentrated exosome-like vesicles that are applied to target cells prior to infection with virus. ooster treatment increases the cell surface density of the receptor recognized by the infecting virus, thus increasing transduction efficiency. Using this technology, for example, you can coat human cells with the ecotropic receptor (which is otherwise absent), enabling them to be transduced with ecotropic retrovirus or ecotropic lentivirus. Ecotropic Receptor ooster human 세포에 ecotropic pseudotyped 렌 티바이러스또는레트로바이러스감염 Viral Receptor oosters 를이용하면이전에감염이어려웠던세포에 서바이러스의형질도입이가능하다. 예를들면, human 세포의경 우일반적으로 mcat-1 receptor 이발현되지않기때문에 ecotropicpseudotyped 렌티바이러스또는레트로바이러스를이용하여형질도입 이어렵다. 하지만 human cell 에 ecotropic pseudotyped 렌티바이러스 를형질도입시키기전에 Ecotropic Receptor ooster 에미리처리를 하면 ecotropic lentivirus 를이용하여 human 세포에형질도입이가능 하다 ( 그림 15, 그림 16).. Receptor oosters are concentrated receptors on exosome-like particles 4. ooster treatment increases infection efficiency 4 Life Science & iotechnology 5

provides the critical 3 to 5' exonuclease or "editing" function that corrects misincor Ecotropic Receptor ooster Infect human cells with ecotropic pseudotyped lentivirus or retrovirus NOTE: The

Using LD&PCR range of possible PCR products to about 6 kb. Viral Receptor oosters transiently confer a viral susceptibility phenotype to previously non-infectible cells.

However, pretreating cells with Ecotropic Receptor ooster allows them to be transduced with ecotropic pseudotyped lentivirus.

neural progenitor cells (Figures and 3). Receptor ooster Lentivirus Lentivirus + Receptor ooster Fluorescence Phase Negative Figure.

HT18 cells were seeded in 6-well plates 4 hr prior to transduction and incubated with 1 µl Ecotropic Receptor ooster for hr. Cells were then 그림 15.

dpackaging u c TSystem i o n(cat. No. 63151) and assayed 48 hr later for ZsGreen1 expression. Ecotropic Receptor ooster treatment.

Cells Obtain equivalent transduction efficiency across target cell types were thenalthough transduced with Lenti-X ZsGreen1 lentivirus (MOI=~15) produced using Clonother factors can also affect viral

normalization transduction efficiency cell lines. human cell lines showed high ZsGreen1transduction expression.

A Human mesenchymal stem cells (hmscs) Normal human neural progenitor cells (NHNPs) 1 % Transduced 8 T r a n s d u c T i o n Fluorescence ficiency across target cell types 6 4 t viral infection, we

Six different human cell lines showed high c lentivirus following treatment with Ecotropic Receptor ooster (Figures chymal MSCs) at SC Ju rk hm pg La 1 8 HT 9 3T V i r a l Phase Jurkat cells Normal

Usually, it is not possible to infect Jurkat progenitor cells (NHNPs) cells, hmscs, or1 NHNPs with ecotropic pseudotyped lentivirus.

A panel of human cell lines, normally resistant to transduction by ecotropicpseudotyped lentiviral vectors, were efficiently transduced after treatment with Ecotropic Receptor ooster.

retrovirus. However, pretreatment with Amphotropic Receptor ooster supplies the active receptor to the surface of these cells for viral infection by the virus (Figure 4). Figure 1.

Flow Chart of the Lenti-X Inteqratin Analysis protocol libraries, PCR amplification of genomic DNA from the libraries, and analysis of PCR products. The gel shows HpaI library. Lane : DraI library.

6 provides the critical 3 to 5' exonuclease or "editing" function that corrects misincor Ecotropic Receptor ooster Infect human cells with ecotropic pseudotyped lentivirus or retrovirus NOTE: The Lenti-X Integration Site Analysis protocol is optimized for Advantag Systems Tools in the we do not recommend using any other enzyme with thislentivirus kit. Using LD&PCR range of possible PCR products to about 6 kb. Viral Receptor oosters transiently confer a viral susceptibility phenotype to previously non-infectible cells. For example, human cells are not normally infected by ecotropic-pseudotyped lentivirus or retrovirus, since they do not express the mcat-1 receptor. However, pretreating cells with Ecotropic Receptor ooster allows them to be transduced with ecotropic pseudotyped lentivirus. Human cells that have been efficiently transduced with ecotropic lentivirus after Ecotropic Receptor ooster include: HT18, HEK93T, La, pg, Jurkat, human mesenchymal stem cells, and normal human neural progenitor cells (Figures and 3). Receptor ooster Lentivirus Lentivirus + Receptor ooster Fluorescence Phase Negative Figure. Viral transduction of human cells with ecotropic lentivirus following Ecotropic Receptor ooster treatment. HT18 cells were seeded in 6-well plates 4 hr prior to transduction and incubated with 1 µl Ecotropic Receptor ooster for hr. Cells were then 그림 15. of human cells with ecotropic lentivirus following transduced withviral Lenti-X transduction ZsGreen1 lentivirus (MOI=~15) produced using Clontech s V i r a Lenti-X l T HTX r Ecotropic a n s dpackaging u c TSystem i o n(cat. No ) and assayed 48 hr later for ZsGreen1 expression. Ecotropic Receptor ooster treatment. HT18 cells were seeded in 6-well plates 4 hr prior to transduction and incubated with 1 μl Ecotropic Receptor ooster for hr. Cells Obtain equivalent transduction efficiency across target cell types were thenalthough transduced with Lenti-X ZsGreen1 lentivirus (MOI=~15) produced using Clonother factors can also affect viral infection, we have observed that receptor booster treatment leads to tech s Lenti-X Ecotropic of Packaging System (Code across 63151) multiple and assayed 48 hr Six laterdifferent for near HTX normalization transduction efficiency cell lines. human cell lines showed high ZsGreen1transduction expression. efficiencies for ecotropic lentivirus following treatment with Ecotropic Receptor ooster (Figures and 3). A Human mesenchymal stem cells (hmscs) Normal human neural progenitor cells (NHNPs) 1 % Transduced 8 T r a n s d u c T i o n Fluorescence ficiency across target cell types 6 4 t viral infection, we have observed that receptor booster treatment leads to ficiency across multiple cell lines. Six different human cell lines showed high c lentivirus following treatment with Ecotropic Receptor ooster (Figures chymal MSCs) at SC Ju rk hm pg La 1 8 HT 9 3T V i r a l Phase Jurkat cells Normal human neural Figure 3. Ecotropic Receptor ooster aides transduction of ecotropic lentivirus. Panel A. Usually, it is not possible to infect Jurkat progenitor cells (NHNPs) cells, hmscs, or1 NHNPs with ecotropic pseudotyped lentivirus. However, pre-treatment of these cell types with Ecotropic Receptor % Transduced ooster allows for very efficient transduction. Panel. A panel of human cell lines, normally resistant to transduction by ecotropicpseudotyped lentiviral vectors, were efficiently transduced after treatment with Ecotropic Receptor ooster. 8 6 Amphotropic4Receptor ooster For efficient transduction of amphotropic pseudotyped retrovirus Chinese hamster ovary (CHO) cells are typically inefficiently transduced with amphotropic-pseudotyped retrovirus. However, pretreatment with Amphotropic Receptor ooster supplies the active receptor to the surface of these cells for viral infection by the virus (Figure 4). Figure 1. Flow chart of the그림 Lenti-X Integration Site Analysis protocol. The major steps include constructi 17. Flow Chart of the Lenti-X Inteqratin Analysis protocol libraries, PCR amplification of genomic DNA from the libraries, and analysis of PCR products. The gel shows HpaI library. Lane : DraI library. Lane 3: SspI library. Lane 4: No template control. Lane M: DNA size mark 8 transduction of ecotropic lentivirus. Panel A. Usually, it is not possible to infect Jurkat LSP: Lentivirus-specific primer. 제품 리스트 16. Ecotropic transduction of ecotropic lentivirus. udotyped lentivirus.그림 However, pre-treatmentreceptor of these cellooster types withaides Ecotropic Receptor at Ju rk SC 1 8 pg hm HT La % Transduced T n. Panel. A panelpanel of human cell lines,itnormally resistant to by ecotropica. Usually, is not possible totransduction infect Jurkat cells, hmscs, or NHNPs with ecotropic ntly transduced after treatment with Ecotropic Receptor ooster. pseudotyped lentivirus. However, pre-treatment of these cell types with Ecotropic Re4 ceptor ooster allows for very efficient transduction. Panel. A panel of human cell lines, (4413) normally resistant to transduction by ecotropicpseudotyped lentiviral vectors, were effifor efficient transduction of amphotropic pseudotyped retrovirus ciently transduced after treatment with Ecotropic Receptor ooster. Code 제품명 Lenti-X Expression System Lenti-X Expression System (EF1alpha) Clontech Laboratories, A Takara Lenti-X icistronic ExpressionInc. System (Neo) io Company Lenti-X Tet-On 3G Inducible Expression System e typically inefficiently transduced with amphotropic-pseudotyped retrovirus. Negative ooster ooster + ooster + virus (MOI 5.6) virus (MOI 56.4) ropic Receptor ooster supplies the active receptor to the surface(moi of 5.6) these (MOI 56.4) Lenti-X Tet-Express Inducible Expression System TM igure 4). Lenti-XFigure Integration Siteooster Analysis Kit efficient transduction of CHO-K1 cells. CHO-K1 cells were plated at 1.5 x 15 cells/ 4. Amphotropic Receptor treatment enables Lenti-X shrna Expression System well on a 6-well plate. The next day, the cells were treated with Amphotropic Receptor ooster and transduced with amphotropic retrovirus Lenti-X HTX Packaging System expressing ZsGreen1 fluorescent protein. The cells were analyzed for fluorescence by flow cytometry hours post-transduction. 회. GenomeWalker를 기반으로 provirus의 게놈 삽입 부위를 식별 Lenti-X HTX Ecotropic Packaging System 회. 대부분의 렌티바이러스 벡터에 적용 가능 Lenti-X HTX Packaging System (Integrase Deficient) 회 6318 Lenti-X 93T Cell 3 Line Lenti-X Concentrator Lenti-X Maxi Purification Kit Lenti-X GoStix 부분을 정확하게 분석할 수 있다. 렌티바이러스는 RNA genome virus Lenti-X qrt-pcr Titration Kit 회 로서 DNA로 역전사 되어 host genome에 삽입된다. provirus는 host Lenti-X Provirus Quantitation Kit 회 virus (MOI 5.6) virus (MOI 56.4) genome에 무작위로 삽입되기 때문에 목적유전자 및 endogenous 유 63 Lenti-X p4 Rapid Titer Ki 96 회 Ecotropic Receptor ooster 회 Lenti-X Integration Site Analysis Kit Lenti-X Integration Site Analysis Kit는 GenomeWalker 기술을 기 반으로 렌티바이러스 벡터가 host genome DNA에 provirus로 삽입된 (MOI 5.6) 용량 (MOI 56.4) ooster ooster + ooster + ment enables efficient transduction of CHO-K1 cells. CHO-K1 cells were plated at 1.5 x 15 cells/ 전자 발현에 영향을 줄 수 있다. 따라서 provirus의 were treated with Amphotropic Receptor ooster and transduced with amphotropic retrovirus he cells were analyzed for cell의 fluorescence by flow연구에 cytometry중요하다. 48 hours post-transduction. host 표현형 3 삽입부위의 식별은 1 1 preps 회 License Notice [K11, K19, K1, K5, K7, K9, K31, K38, K4, K45, K47, K6, K69, K7] 5

스생산은최적화된시스템구성성분들간의시너지효과에의한것이다 ( 그림 1). 첫째, 클론텍의새로운 Lenti-X HT Packaging Mix는 Gag-Pr, Tat, Rev, RT, IN과 VSV-G prteins을높은레벨로발현하는벡터에의해필수적인렌티바이러스패키징구성요소를완

스생산은최적화된시스템구성성분들간의시너지효과에의한것이다 ( 그림 1). 첫째, 클론텍의새로운 Lenti-X HT Packaging Mix는 Gag-Pr, Tat, Rev, RT, IN과 VSV-G prteins을높은레벨로발현하는벡터에의해필수적인렌티바이러스패키징구성요소를완 Lentiviral delivery system 에관한모든것은클론텍에서 Lenti-X TM Expressin System [1] 클론텍의 Viral Gene Delivery Systems 클론텍은어떤세포에도적용할수있는모든바 이러스시스템을갖추고있다. Lentiviral 이나 retrviral 또는 adenviral systems 중에서선택가능 모든시스템이고발현구조를갖고있으며