Adam M A, Osborne W R, Miller A D
Department of Biochemistry and Molecular Biology, Merck Frosst Canada, Inc., Kirkland, Quebec, Canada.
Hum Gene Ther. 1995 Sep;6(9):1169-76. doi: 10.1089/hum.1995.6.9-1169.
Protein expression from retroviral vectors is often highest when the expressed cDNA is driven by the retroviral promoter. However, the typical retroviral vector design places the cDNA downstream of the retroviral packaging signal and far from the retroviral promoter. In an attempt to improve protein production levels from cDNAs expressed in retroviral vectors, we inserted the MyoD or the purine nucleoside phosphorylase (PNP) cDNAs into the R regions of both retroviral LTRs, close to the retroviral promoter and just upstream of the polyadenylation signal present in each long terminal repeat (LTR). These R-region double-copy vectors could be produced in unrearranged form, although the titer was about seven-fold lower than that of typical vectors. R-region positioning of the MyoD cDNA resulted in five-fold higher MyoD expression compared to MyoD expression in a typical vector, whereas PNP expression was not improved. Thus, R-region double-copy vectors provide an alternative vector design that can improve protein expression from some cDNAs.
当表达的cDNA由逆转录病毒启动子驱动时，逆转录病毒载体的蛋白质表达通常最高。然而，典型的逆转录病毒载体设计将cDNA置于逆转录病毒包装信号的下游，且远离逆转录病毒启动子。为了提高逆转录病毒载体中表达的cDNA的蛋白质产生水平，我们将MyoD或嘌呤核苷磷酸化酶（PNP）cDNA插入到两个逆转录病毒LTR的R区域，靠近逆转录病毒启动子且恰好在每个长末端重复序列（LTR）中存在的聚腺苷酸化信号的上游。这些R区域双拷贝载体可以以未重排的形式产生，尽管其滴度比典型载体低约7倍。与典型载体中的MyoD表达相比，MyoD cDNA在R区域的定位导致MyoD表达提高了5倍，而PNP表达没有改善。因此，R区域双拷贝载体提供了一种替代的载体设计，可以提高某些cDNA的蛋白质表达。
