White M R, Masuko M, Amet L, Elliott G, Braddock M, Kingsman A J, Kingsman S M
Amersham International, Bucks, UK.
J Cell Sci. 1995 Feb;108 ( Pt 2):441-55. doi: 10.1242/jcs.108.2.441.
The regulation of human cytomegalovirus (hCMV) and human immunodeficiency virus (HIV) gene expression has been studied in single intact mammalian cells. Viral promoters were placed upstream of the firefly luciferase reporter gene and the resulting hybrid reporter constructs were stably integrated into the HeLa cell genome. A highly sensitive photon-counting camera system was used to study the level of gene expression in single intact cells. Luciferase expression was studied in the absence of activators of viral gene expression, in the presence of the HIV-1 TAT transactivator protein, or in the presence of sodium butyrate, a non-viral activator of gene expression. In the absence of any activator of gene expression, while expression was undetectable in most cells, significant levels of basal luciferase activity were observed in a few cells, indicating heterogeneity in gene expression in the cell population. In the presence of the general activator of viral gene expression, sodium butyrate, transcriptional activation from the viral promoters gave rise to significant and relatively homogeneous levels of luciferase expression in a majority of cells. The luciferase imaging technology was used for the real-time analysis of changes of gene expression within a single cell. This non-invasive reporter assay should become important for studies of the temporal regulation of gene expression in single cells.
人类巨细胞病毒（hCMV）和人类免疫缺陷病毒（HIV）基因表达的调控已在单个完整的哺乳动物细胞中进行了研究。将病毒启动子置于萤火虫荧光素酶报告基因的上游，并将所得的杂交报告构建体稳定整合到HeLa细胞基因组中。使用高灵敏度的光子计数相机系统研究单个完整细胞中的基因表达水平。在不存在病毒基因表达激活剂的情况下、在存在HIV-1 TAT反式激活蛋白的情况下或在存在丁酸钠（一种基因表达的非病毒激活剂）的情况下研究荧光素酶表达。在不存在任何基因表达激活剂的情况下，虽然在大多数细胞中检测不到表达，但在少数细胞中观察到了显著水平的基础荧光素酶活性，这表明细胞群体中基因表达存在异质性。在存在病毒基因表达的通用激活剂丁酸钠的情况下，病毒启动子的转录激活在大多数细胞中产生了显著且相对均匀的荧光素酶表达水平。荧光素酶成像技术用于实时分析单个细胞内基因表达的变化。这种非侵入性报告分析对于研究单个细胞中基因表达的时间调控应该会变得很重要。
