FLUORESCENCE-ACTIVATED CELL ANALYSIS AND SORTING OF VIABLE MAMMALIAN-CELLS BASED ON BETA-D-GALACTOSIDASE ACTIVITY AFTER TRANSDUCTION OF ESCHERICHIA-COLI LACZ
FLUORESCENCE-ACTIVATED CELL ANALYSIS AND SORTING OF VIABLE MAMMALIAN-CELLS BASED ON BETA-D-GALACTOSIDASE ACTIVITY AFTER TRANSDUCTION OF ESCHERICHIA-COLI LACZ
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DOI：
10.1073/pnas.85.8.2603
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发表时间：
1988-04-01
影响因子：
11.1
通讯作者：
HERZENBERG, LA
中科院分区：
文献类型：
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作者：
NOLAN, GP;FIERING, S;HERZENBERG, LA
我们证明，感染并表达携带大肠杆菌β-半乳糖苷酶基因(lacZ)的重组逆转录病毒的个体细胞可以通过基于lacZ表达水平的荧光激活细胞分选进行活染色、分析、分选和克隆。为了实现这一目标，我们设计了一种在活哺乳动物细胞中酶促产生和维持荧光的方法。细胞中荧光产物的积累与时间呈线性关系，荧光与酶活性直接相关。这种用于β-半乳糖苷酶检测的技术比其他可用的细胞化学或生物化学方法更灵敏。我们已经使用该程序来显示T细胞淋巴瘤BW5147和B细胞杂交瘤SP2/0中的.psi.-2-MMuLVSVnlsLacZ的表达并不完全稳定，并且通过荧光激活细胞分选仪选择的低lacZ活性的亚克隆表现出明显较低的LacZ平均表达。这些发现表明β-半乳糖苷酶作为单细胞水平的报告分子用于基因调控研究的效用，包括启动子功效、增强子活性、反式作用因子和其他调控元件的研究。
We demonstrate that individual cells infected with and expressing a recombinant retrovirus carrying the Escherichia coli .beta.-galactosidase gene (lacZ) can be viably stained, analyzed, sorted, and cloned by fluorescence-activated cell sorting based on the levels of lacZ expressed. To accomplish this we have devised a method to enzymatically generate and maintain fluorescence in live mammalian cells. Accumulation of fluorescent products in cells is linear with time, with a direct correlation of fluorescence to enzymatic activity. This technology for .beta.-galactosidase detection is more sensitive than other available cytochemical or biochemical methods. We have used this procedure to show that the expression of .psi.-2-MMuLVSVnlsLacZ in the T-cell lymphoma BW5147 and the B-cell hybridoma SP2/0 is not completely stable and that subclones selected by the fluorescence-activated cell sorter for low lacZ activity demonstrate distinctly lower average expression of LacZ. These findings indicate the utility of .beta.-galactosidase as a reporter molecule at the single-cell level for studies of gene regulation, including studies of promoter efficacy, enhancer activity, trans-acting factors, and other regulatory elements.