Design of a retrovirus-derived vector for expression and transduction of exogenous genes in mammalian cells.
We have developed a transfection vector for animal cells that contains long terminal repeat (LTR) sequences to promote expression. Plasmid p101/101, a derivative of plasmid pBR322 containing the complete Moloney murine sarcoma virus genome, was cut with restriction enzymes and religated so that both the 5' and 3' LTRs were retained and all but about 700 base pairs of the intervening viral sequences were removed. To test this vector, the Escherichia coli gene gpt was cloned into a unique PstI site, between the two LTRs, with guanine and cytosine tailing, a method that can be generalized for insertion of any DNA segment into this vector. When DNA from recombinant plasmids in which the gpt gene was inserted in the same transcriptional polarity as the LTR sequences was transfected onto murine or rat fibroblast cultures, we obtained a high yield of Gpt(+) colonies. However, plasmid constructs with the gpt gene in the opposite polarity were virtually devoid of activity. With gpt in the proper orientation, restriction enzyme cuts within the LTRs or between the 5' LTR and the gpt gene reduced transfection by more than 98%, whereas a cut between the gpt gene and the 3' LTR gave an 80% reduction in activity. Thus, both 5' and 3' LTR sequences are essential for optimal gpt expression, although the 5' LTR appears to play a more important role. When the LTR-gpt plasmid was transfected onto murine leukemia virus-infected mouse fibroblasts, we obtained evidence that RNA copies became pseudotyped into viral particles which could transfer the Gpt(+) phenotype into rodent cells with extremely high efficiency. This vector should prove useful for high-efficiency transduction of a variety of genes in mammalian cells.
D000818:Animals; D004262:DNA Restriction Enzymes; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006378:Helper Viruses / Q000235:genetics; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics; D010430:Pentosyltransferases / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D014158:Transcription, Genetic; D014161:Transduction, Genetic
D010430:Pentosyltransferases; C024827:xanthine phosphoribosyltransferase; D004262:DNA Restriction Enzymes