Retroviral vectors encoding a reverse transcription-activated transgene efficiently limit expression of the gene to target cells.
Recombinant retroviral vectors are indispensable tools for the study of gene function and for therapeutic gene transfer owing to their ability to transfer and stably express foreign genes in target cells. A limitation of these vectors, however, is the difficulty in generating stable vector producer cell (VPC) lines when the vectors encode cytotoxic proteins. We developed a series of Moloney murine leukemia virus-based vectors encoding a reverse transcription-activated transgene. These vectors preclude gene expression in the producer cells, yet allow lines for transgene expression in target cells. The vectors were generated by cloning the gene of interest in reverse orientation either just upstream of the viral 3' long terminal repeat (LTR) or in the U3 region of the 3'LTR. An exogenous promoter was inserted, also in reverse orientation, at the R-U5 border of the viral 5'LTR. Upon transduction of target cells, the inserted promoter is copied to the 3'LTR during reverse transcription of the vector genomic RNA, where it then drives transgene expression. We tested this system using a green fluorescent protein (GFP) gene and the SV40 promoter. Reverse transcription-activated retroviral vectors may allow for the generation of stable retroviral VPC lines encoding cytotoxic or inhibitory genes.
Burke
John Douglas
JD
Metabolism Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
Morris
John C
JC
D002460:Cell Line; D015870:Gene Expression / Q000235:genetics*; D020868:Gene Silencing; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D048348:Reverse Transcription / Q000235:genetics*; D019076:Transgenes / Q000235:genetics*
D012333:RNA, Messenger