IngentaConnect Bicistronic and Two-Gene Retroviral Vectors for Using MDR 1 as a ...

Abstract:

We describe a series of two-gene and bicistronic retroviral vectors that use the human MDR 1 gene as a selectable marker for the overexpression of a second heterologous gene in transduced cells. The vectors use Harvey murine sarcoma virus sequences for viral expression and packaging functions and include sites for cloning foreign genes of interest under the control of either an internal promoter (two-gene vectors) or an internal ribosome entry site (bicistronic vectors). To characterize these vectors, we used neo as a reporter gene for foreign gene expression and as an independently selectable marker for comparison with MDR 1. Each of the vector constructions supported high-titer retrovirus production and transduction of mouse and human cell lines. Using MDR 1- neo virus supernatants in parallel titering assays, we found that titers based on colchicine resistance were 10- to 20-fold lower than titers based on G418 resistance, suggesting that MDR 1 is a more stringent selectable marker than neo in NIH 3T3 and KB-3-1 cell lines. Whereas neo gene expression with the two-gene vectors was subject to host-specific limitations on internal promoter activity, the bicistronic vectors were highly active in three cell lines tested. In K562 cells, using the bicistronic vector, selection with colchicine led to at least 20-fold higher expression of the MDR 1 gene product than did selection with G418, suggesting that the stringent MDR 1 selection system is very efficient for obtaining overexpression of foreign genes. Retroviral vectors carrying MDR 1 as a selectable marker plus a second, heterologous gene of interest could have widespread utility for in vitro and in vivo applications of gene transfer technology, including gene therapy.

Language: English

Document Type: Research article

Affiliations: 1: Department of Cell and Tumor Biology, City of Hope National Medical Center, 1500 E. Duarte Road, Duarte, California, 91010 2: Medical Oncology and Therapeutics Research, City of Hope National Medical Center, 1500 E. Duarte Road, Duarte, California, 91010

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Bicistronic and Two-Gene Retroviral Vectors for Using MDR 1 as a Selectable Marker and a Therapeutic Gene