Edamatsu H, Kaziro Y, Itoh H
Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama, Japan.
Gene. 1997 Mar 18;187(2):289-94. doi: 10.1016/s0378-1119(96)00768-8.
We have constructed an inducible high-level expression vector, pEF-LAC. pEF-LAC has a modified human polypeptide chain elongation factor 1alpha (EF-1alpha) promoter containing three lactose operator sequences. Using the cat reporter gene, we characterized the transcriptional activity of pEF-LAC. In the transient transfection of NIH3T3 and BaF3 cells, the transcriptional activity of pEF-LAC was higher than that of the original human elongation factor 1alpha promoter, simian virus 40 (SV40) promoter, and Rous sarcoma virus (RSV) long terminal repeat (LTR). Cotransfection of the lactose repressor expression plasmid effectively suppressed the promoter activity of pEF-LAC, and the activity was fully recovered by addition of isopropyl beta-D-thiogalactopyranoside (IPTG). Even in the stable transfection of Rat-1 cells, the promoter activity of the integrated pEF-LAC was much higher than that of the RSV-LTR and regulated in an IPTG-dependent manner. These results suggest that pEF-LAC is a useful vector for the inducible high-level expression of the cloned gene in a variety of mammalian cells.
我们构建了一种可诱导的高效表达载体pEF-LAC。pEF-LAC具有一个经过修饰的人多肽链延长因子1α（EF-1α）启动子，其中包含三个乳糖操纵序列。利用氯霉素乙酰转移酶（cat）报告基因，我们对pEF-LAC的转录活性进行了表征。在NIH3T3和BaF3细胞的瞬时转染中，pEF-LAC的转录活性高于原始的人延长因子1α启动子、猿猴病毒40（SV40）启动子和劳氏肉瘤病毒（RSV）长末端重复序列（LTR）。乳糖阻遏物表达质粒的共转染有效地抑制了pEF-LAC的启动子活性，而通过添加异丙基β-D-硫代半乳糖苷（IPTG）可使活性完全恢复。即使在Rat-1细胞的稳定转染中，整合的pEF-LAC的启动子活性也远高于RSV-LTR，并且以IPTG依赖的方式受到调控。这些结果表明，pEF-LAC是一种用于在多种哺乳动物细胞中诱导克隆基因高效表达的有用载体。
