IMPROVED FACS-GAL - FLOW CYTOMETRIC ANALYSIS AND SORTING OF VIABLE EUKARYOTIC CELLS EXPRESSING REPORTER GENE CONSTRUCTS
IMPROVED FACS-GAL - FLOW CYTOMETRIC ANALYSIS AND SORTING OF VIABLE EUKARYOTIC CELLS EXPRESSING REPORTER GENE CONSTRUCTS
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DOI：
10.1002/cyto.990120402
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发表时间：
1991-01-01
期刊：
影响因子：
--
通讯作者：
HERZENBERG, LA
中科院分区：
文献类型：
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作者：
FIERING, SN;ROEDERER, M;HERZENBERG, LA
先前报道的FACS-Gal测定（Nolan等人，Proc Natl Acad Sci USA 85：2603-2607，1988）测量E.大肠杆菌lacZ编码的β-半乳糖苷酶活性在单个活的真核细胞中用于多种分子和细胞生物学应用。 酶活性通过流式细胞术测量，使用荧光底物，其被水解并保留在细胞内。 在该系统中，lacZ既作为报告基因来定量基因表达，又作为选择性标记，用于基于细胞的lacZ表达水平对细胞进行荧光激活分选。 本报告详细介绍了原始测定的以下改进：1）使用苯乙基-β-D-硫代半乳糖苷（一种竞争性抑制剂）抑制β-半乳糖苷酶活性; 2）通过双色测量减少假阳性; 3）通过弱碱性氯喹抑制干扰哺乳动物β-半乳糖苷酶。 我们发现在该测定中由β-半乳糖苷酶产生的荧光与β-半乳糖苷酶分子的细胞内浓度之间存在指数关系。 最后，我们报告的最佳装载的基板（FDG）和保留的产品，荧光素的条件。 在这些条件下，我们发现在单个实验中，FDG在克隆的所有细胞中均匀加载。 总之，这些改进使得FACS-Gal成为研究真核细胞中基因表达的非常强大的工具。
The previously reported FACS-Gal assay (Nolan et al., Proc Natl Acad Sci USA 85:2603-2607, 1988) measures E. coli lacZ-encoded beta-galactosidase activity in individual viable eukaryotic cells for a variety of molecular and cellular biological applications. Enzyme activity is measured by flow cytometry, using a fluorogenic substrate, which is hydrolyzed and retained intracellularly. In this system, lacZ serves both as a reporter gene to quantitate gene expression and as a selectable marker for the fluorescence-activated sorting of cells based on their lacZ expression level. This report details the following improvements of the original assay: 1) use of phenylethyl-beta-D-thiogalactoside, a competitive inhibitor, to inhibit beta-galactosidase activity; 2) reduction of false positives by two-color measurements; and 3) inhibition of interfering mammalian beta-galactosidases by the weak base chloroquine. We found an exponential relationship between fluorescence generated by beta-galactosidase in this assay and the intracellular concentration of beta-galactosidase molecules. Finally, we report conditions for optimal loading of the substrate (FDG) and retention of the product, fluorescein. Under these conditions, we found uniform loading of FDG in all cells of a clone in individual experiments. Together, these improvements make FACS-Gal an extremely powerful tool for investigation of gene expression in eukaryotic cells.