Liu X, Giza C C, Vrana K E
West Virginia University Health Sciences Center.
Biotechniques. 1990 Aug;9(2):168-73.
We have compared the transcriptional efficiencies of a number of eukaryotic promoters following DNA-mediated transfection into cultured rat hepatoma cells. We find that the highest levels of expression for the bacterial chloramphenicol acetyltransferase (CAT) reporter gene are observed with a herpes simplex virus type 1 (HSV-1) immediate early promoter when co-transfected with an expression construct bearing the gene for the HSV-1 transcriptional activator protein VP16. This transactivation phenomenon is specific for the HSV-1 immediate early promoter and increases the expression of the reporter gene 7-fold. Expression from the ICP4 promoter is 2.5-fold greater than the other promoters tested. In addition, expression from the ICP4 promoter can be induced, at varying times following transfection, by infecting the cells with HSV-1 viral particles. Two plasmids have been constructed which contain the HSV-1 ICP4 promoter adjacent to a multiple cloning site. One of the plasmids also contains SV40 splicing and polyadenylation signals.
我们比较了多种真核启动子在经DNA介导转染入培养的大鼠肝癌细胞后的转录效率。我们发现，当与携带单纯疱疹病毒1型（HSV-1）转录激活蛋白VP16基因的表达构建体共转染时，使用单纯疱疹病毒1型（HSV-1）立即早期启动子可观察到细菌氯霉素乙酰转移酶（CAT）报告基因的最高表达水平。这种反式激活现象对HSV-1立即早期启动子具有特异性，可使报告基因的表达增加7倍。来自ICP4启动子的表达比其他测试启动子高2.5倍。此外，在转染后的不同时间，通过用HSV-1病毒颗粒感染细胞，可诱导ICP4启动子的表达。构建了两个质粒，它们含有与多克隆位点相邻的HSV-1 ICP4启动子。其中一个质粒还含有SV40剪接和聚腺苷酸化信号。
