Use of recombinant DNA technology to program eukaryotic cells to synthesize rat proinsulin: a rapid expression assay for cloned genes

Abstract

To use recombinant DNA technology to functionally analyze mutations introduced into cloned eukaryotic genes, a rapid procedure is necessary to assay the steps along the gene expression pathway. Since cloned rat insulin genes are not transcribed efficiently after transfection into various cell lines, I have asked whether one could drive expression by placing the insulin gene inside a transcriptional unit that functions in all mammalian cells. By using a small simian virus 40 (SV40) fragment that contains initiation signals for replication and transcription, I connected the 5'-noncoding region of the SV40 tumor antigen gene to the 5'-noncoding region of the rat insulin II gene to create a pBR322-based recombinant. If one assays shortly after its introduction into mammalian cells, it can be shown that this recombinant plasmid programs the synthesis of correctly spliced and polyadenylylated insulin mRNA that functions in the synthesis and secretion of rat proinsulin. This system permits rapid analysis of cloned in vitro-engineered mutations and the programming of eukaryotic cells to manufacture proteins that they normally do not synthesize.