Self-amplifying expression from the T7 promoter in 3T3 mouse fibroblasts

Abstract

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-Gl plasmid increases the usefulness of the T7 gene system for expression within mammalian cells.