Deng H, Wolff J A
Department of Pediatrics, Waisman Center, Madison, WI 53705.
Gene. 1994 Jun 10;143(2):245-9. doi: 10.1016/0378-1119(94)90104-x.
To increase the levels of exogenous or foreign gene expression in mammalian cells, this study sought to develop an 'autogene' that will self-amplify. An autogene plasmid, pT7-G1, containing the T7 phage RNA polymerase-encoding modified gene (G1) under control of its cognate T7 promoter, was only obtained when the plasmid contained the encephalomyocarditis (EMC) untranslated sequence. In vitro transcription and translation studies confirmed that both the T7 promoter and the G1 gene were completely functional in the pT7-G1 plasmid. Expression from pT7-G1 was initiated in vivo either by co-transfection with its in vitro transcript or by transfection into NIH3T3 cell lines that stably expressed T7 RNA polymerase enzyme. Use of the pT7-G1 autogene enabled an approx. 50-fold increase in foreign protein production. Northern analysis suggested that this increased expression resulted from the self-amplification of the autogene. By allowing greater expression in cell lines with low T7 RNA polymerase expression, the pT7-G1 plasmid increases the usefulness of the T7 gene system for expression within mammalian cells.
为了提高哺乳动物细胞中外源基因或外源基因的表达水平，本研究试图开发一种能够自我扩增的“自基因”。一种自基因质粒pT7-G1，其在同源T7启动子的控制下含有编码T7噬菌体RNA聚合酶的修饰基因（G1），只有当该质粒含有脑心肌炎病毒（EMC）非翻译序列时才能获得。体外转录和翻译研究证实，T7启动子和G1基因在pT7-G1质粒中均完全具有功能。pT7-G1的表达可以通过与其体外转录本共转染，或者通过转染到稳定表达T7 RNA聚合酶的NIH3T3细胞系中在体内启动。使用pT7-G1自基因能够使外源蛋白产量提高约50倍。Northern分析表明，这种表达增加是由于自基因的自我扩增所致。通过在T7 RNA聚合酶表达水平较低的细胞系中实现更高的表达，pT7-G1质粒提高了T7基因系统在哺乳动物细胞内表达的实用性。
