Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter.
Gene expression was visualized in single living mammalian cells with beta-lactamase as a reporter that hydrolyzes a substrate loaded intracellularly as a membrane-permeant ester. Each enzyme molecule changed the fluorescence of many substrate molecules from green to blue by disrupting resonance energy transfer. This wavelength shift was detectable by eye or color film in individual cells containing less than 100 beta-lactamase molecules. The robust change in emission ratio reveals quantitative heterogeneity in real-time gene expression, enables clonal selection by flow cytometry, and forms a basis for high-throughput screening of pharmaceutical candidate drugs in living mammalian cells.
Zlokarnik
G
G
Aurora Biosciences, 11010 Torreyana Road, San Diego, CA 92121, USA.
Negulescu
P A
PA
Knapp
T E
TE
Mere
L
L
Burres
N
N
Feng
L
L
Whitney
M
M
Roemer
K
K
Tsien
R Y
RY
D000818:Animals; D002460:Cell Line; D002469:Cell Separation / Q000379:methods; D002999:Clone Cells / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D004735:Energy Transfer; D005434:Flow Cytometry; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D015870:Gene Expression; D017930:Genes, Reporter; D006207:Half-Life; D006801:Humans; D007769:Lactams; D018721:Muscarinic Agonists / Q000494:pharmacology; D018727:Muscarinic Antagonists / Q000494:pharmacology; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D014468:Umbelliferones / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism; D047090:beta-Lactams / Q000378:metabolism
C109764:CCF 2; D004268:DNA-Binding Proteins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007769:Lactams; D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D014157:Transcription Factors; D014468:Umbelliferones; D047090:beta-Lactams; C031477:7-hydroxycoumarin; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1