Efficient expression of drug-selectable genes in retroviral vectors under control of an internal ribosome entry site.
We describe a new retroviral vector system pSXLC/pHa that utilizes a putative internal ribosome entry site (IRES) from encephalomyocarditis virus downstream from a multicloning site to co-express drug-selectable markers with a second non-selectable cDNA in a eukaryotic expression vector. The positive drug-selectable marker, MDR1, and the positive-negative marker, herpes simplex virus thymidine kinase (HSV-TK), were successfully introduced and expressed in the pSXLC/pHa system. The pSXLC-MDR and pSXLC-TK vectors contain the drug-selectable genes under translational control of the IRES and multiple cloning sites upstream for insertion of second cDNAs which can be co-expressed in this system. The inserts of these pSXLC plasmids were designed for easy transfer to the pHa retrovirus vector which has a strong promoter from Harvey murine sarcoma virus. The IRES-MDR-carrying retroviral vector, pHa-MCS-IRES-MDR, conferred resistance to vincristine and adriamycin. The IRES-TK-containing vector, pHa-MCS-IRES-TK conferred HAT-resistance in TK-deficient cells and the transfectants showed hypersensitivity to ganciclovir. These "flexible" vectors should be useful for co-expression of genes for selectable gene transfer and for positive-negative (suicide) selections in vitro and in vivo.
Sugimoto
Y
Y
National Cancer Institute, Laboratory of Cell Biology, National Institute of Health, Bethesda, MD 20892.
Aksentijevich
I
I
Gottesman
M M
MM
Pastan
I
I
D016475:3T3 Cells; D000818:Animals; D004262:DNA Restriction Enzymes; D004317:Doxorubicin / Q000494:pharmacology; D004351:Drug Resistance / Q000235:genetics; D015774:Ganciclovir / Q000494:pharmacology; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005819:Genetic Markers; D005822:Genetic Vectors; D051379:Mice; D010957:Plasmids; D012190:Retroviridae / Q000235:genetics*; D012270:Ribosomes / Q000378:metabolism*; D018139:Simplexvirus / Q000201:enzymology; D013937:Thymidine Kinase / Q000235:genetics; D014750:Vincristine / Q000494:pharmacology
D005819:Genetic Markers; D014750:Vincristine; D004317:Doxorubicin; D013937:Thymidine Kinase; D004262:DNA Restriction Enzymes; D015774:Ganciclovir