Tel. 042-860-7028/ Fax.042-861-0307/ E-mail. bigguy@krict.re.kr... 500 3,000.. 40 (genome project).. 1-1. 10 8..,,, / 1, 2, 3 ( 1). 1. 36

35,000 500.. in vitro assay cell-based assay...,.... 1-2... Millennim, Abgenix Medarex.,,,. mrna, DNA,. knockout, knock-in, RNAi... (1).. 1-3...,,,,,,, (mutation). FRET (Fluorescence Resonance Energy Transfer), BRET (Bioluminescence Resonance Energy Transfer) FP (Fluorescence Polarization). HCS (High-content Screening) 96-well, 384-well plate (2). HCS,, Ca++ influx 37

... in vitro ex vivo luminometer....,. (charged coupled device: CCD) (3). 400-1000nm, 2-3 ev CCD, CCD. CCD. CCD HCS. 2-1. (Bioluminescence). (Bioluminescence). (Firefly).. 50,.. 70 (4-7)..,,. (luciferin).. 2-2. (Fluorescence). (Stokes law).. FITC, Texas Red, Cy5, Cy3, DAPI. DAPI,,,. GFP, RFP (8, 9). 38

2-3. (Fluorescence Resonance Energy Transfer; FRET).. FRET 10 ( ) (10).. FRET 1940 Forster, ( 2). FRET. FRET. 1998 G. Zlokarnik β-lactamase CCF2 β-lactamase FRET (11). CCF2/AM β-lactamase FRET 409nm 520nm 447nm. 2. FRET. 2-4. (Bioluminescence Resonance Energy Transfer: BRET) BRET.. FRET (autofluorescence), (photoresponsive), (photobleaching) BRET. BRET (Resonance Energy). BRET FRET (luminescent material) (12)., Perkin Elmer BRET2 Renilla luciferase (Rluc), GFP2, FRET, DeepBlueC., A B, Rluc GFP2 A, B, ( 3). A, B,,,,. assay. BRET, DeepBlueC (DBC) Rluc GFP2 emission, DBC 39

3. BRET (Bioluminescence Resonance Energy Transfer). DBC membrane,,,. 2-5. (Fluorescence Polarization; FP) -,, - DNA,,. (, ). (Anisotropy).. ( 4).,,. FP,. FP,, -.. 4. FP (Fluorescence Polarization) HTS (13)... cell-based assay. in vitro cellbased assay. cell-based assay FITC. (14), (15), (16), β- (17), (18), β- (11), (fluorescent protein) (19), 40

(Secreted placental Alkaline Phosphatase, SEAP) (20).., HCS,. p53 VEGF.,,.. (background activity)...... 7 Kb... mrna..,.. 3-1. Firefly luciferase,,,.,. Photinus Pyralis firefly luciferase (Fluc), 560nm 600nm. Fluc D-luciferin oxyluciferin.,, ATP.. ATP -ATP H 3 PO 4. LH2. LH2+ATP LH2-AMP+2H 3 PO 4 LH2-AMP. AMP (hv). LH2-AMP+1/2 O2 L-AMP*+H2O L-AMP* L+AMP+hv ( ) L, L-AMP* -AMP. LH2-AMP (1/2 O2)., ATP. ATP. 2 Mg2, Mn2, Co2 41

. 1,.. Fluc -, in vivo luciferin. 20., 1. ATP (4). 3-2. Renilla luciferase Renillar reniformis Fluc renilla luciferase (Rluc). coelenterazine. Fluc,, ATP ATP. Fluc - in vivo. coelenterazine. 480 nm, 600 nm (5, 6).. 480nm BRET (Bioluminescence Resonance Energy Transfer). 3-3. Secreted placental alkaline phosphatase (SPAP) SPAP Placental alkaline phosphatase 30. SPAR (21). p-nitrophenol phosphate, CSPD (22). 65 C endogenous alkaline phosphatases. HTS (FBS, Fetal Bovine Serum)... HTS. 3-4. Green fluorescence protein Green fluorescence protein (GFP) Aequorea victoria. cofactor (excitation wavelength). 450-490 nm 510-570 nm.,, GFP. 30 EGFP., ( 5). 42

.. 3-5. Red fluorescence protein (RFP) R. Tsien Discosoma DsRed. 500-550 nm 575-650 nm ( 5). 600nm. DsRed tetramer monomeric red flurescence protein (9)..,,,,, (High Throughput Screening, HTS)., HTS (23). QC,, (% Activity) IC 50, Ki, Km curve- fitting. HTS 5. (Excitation) (Emission)..,, (CCL),. 1990,. HTS,. 1 HTS. :. :. :.. 43

:. 384-well 1536- well ( 6). :.. : HTS 50%.,,. :,,,,. : HTS. :.. HTS,,., (Chemical Inventory),..,.,.,. 6. HTS 96-well.,.. HTS,. HTS,, 1 (, ) 2 ( ). 1 2. 2 1. (HCS), (24). HCS,,. HCS 44

7. HCS, Evotec Opera,,,, -,,,,.,.,,. 21 HTS ( 7). HCS,,,,,,.,,, DNA ( ),,, (device).,., (DNA),.,, DNA Chip ( ), Protein Chip ( ), Cell Chip ( ), Neuron Chip ( ), Chip, Lab-on-a-Chip... 6-1. SPR (surface plasmon resonance) BIOCORE SPR (surface plasmon resonance). SPR.. 180 Da (Dalton)., ( 8). SPR. SPR,,. SPR 1990. HTS SPR, 45

8. SPR Sensorgram 9. caspase 2 SPR SPR image system. SPR sensor system, HTS (25). 6-2. (Chemical chip) 1 (26). ( 9).. Schreiber 3,780 Ure2p, Ure2p Uretupamine (27). 1990.,.. (28).,.,,, 2 3.. 46

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