Petitclerc D, Attal J, Théron M C, Bearzotti M, Bolifraud P, Kann G, Stinnakre M G, Pointu H, Puissant C, Houdebine L M
Agriculture et Agro-Alimentaire Canada, Est Lennoxville, Quebec.
J Biotechnol. 1995 Jun 21;40(3):169-78. doi: 10.1016/0168-1656(95)00047-t.
Various combinations of promoters, introns and transcription terminators were used to drive the expression of bovine growth hormone (bGH) cDNA in different cell types. In constructs containing the human cytomegalovirus (hCMV) promoter and the SV40 late genes terminator, the intron from SV40 genes (VP1) was much more efficient, than the intron from the early genes (t). The synthetic intron SIS generated by the association of an adenovirus splice donor and an immunoglobulin G splice acceptor showed the highest activity. The respective potency of these introns was similar in several mammalian (CHO, HC11 and COS) and fish (TO2 and EPC) cells. The rabbit whey acidic protein (WAP) gene promoter was highly efficient to drive the expression of bGH gene in the HC11 mammary cell lines. In contrast, the bGH cDNA under the control of the same promoter was much less efficiently expressed when the SV40 VP1 intron and transcription terminator were used. The rabbit WAP gene and the human GH gene terminators did not or only moderately enhanced the expression of the construct WAP bGH cDNA. Introduction of a promoter sequence from the mouse mammary tumor virus (MMTV) LTR in the VP1 intron increased very significantly the expression of the WAP bGH cDNA. Although several of these vectors showed high potency when expressed stably in HC11 cells, all of them were only moderately efficient in transgenic mice. These data indicate that the VP1 and the SIS introns may be used to express foreign cDNAs with good efficiency in different cell types. The addition of an enhancer within an intron may still reinforce its efficiency. However, transfection experiments, even when stable expression is carried out, are poorly predictive of the potential efficiency of a vector in transgenic animals.
采用多种启动子、内含子和转录终止子组合，在不同细胞类型中驱动牛生长激素（bGH）cDNA的表达。在含有人类巨细胞病毒（hCMV）启动子和SV40晚期基因终止子的构建体中，来自SV40基因（VP1）的内含子比来自早期基因（t）的内含子效率更高。由腺病毒剪接受体和免疫球蛋白G剪接供体组合产生的合成内含子SIS活性最高。这些内含子在几种哺乳动物（CHO、HC11和COS）和鱼类（TO2和EPC）细胞中的相应效力相似。兔乳清酸性蛋白（WAP）基因启动子在HC11乳腺细胞系中高效驱动bGH基因的表达。相反，当使用SV40 VP1内含子和转录终止子时，相同启动子控制下的bGH cDNA表达效率要低得多。兔WAP基因和人类GH基因终止子未增强或仅适度增强构建体WAP bGH cDNA的表达。在VP1内含子中引入小鼠乳腺肿瘤病毒（MMTV）LTR的启动子序列可非常显著地增加WAP bGH cDNA的表达。尽管其中一些载体在HC11细胞中稳定表达时显示出高效力，但它们在转基因小鼠中均仅具有中等效率。这些数据表明，VP1和SIS内含子可用于在不同细胞类型中高效表达外源cDNA。在内含子中添加增强子可能仍会增强其效率。然而，即使进行稳定表达，转染实验对载体在转基因动物中的潜在效率的预测性也很差。
