Construction and characterization of bicistronic retroviral vectors encoding the multidrug transporter and...

Multidrug resistance (MDR) can be conferred by overexpression of the adenosine triphosphate-driven multidrug transporter P-glycoprotein (Pgp) known as MDR1. Thus, two potential applications of the MDR1 gene that may be useful in gene therapy are the protection of bone marrow cells from the cytotoxic effects of chemotherapy regimens in cancer patients and its possible use as an in vivo selectable gene when linked to a therapeutic gene. In this study, we have designed two retroviral bicistronic expression vectors by linking the MDR1 gene to the reporters known as beta-galactosidase and the red-shifted green fluorescent protein (GFP). We report the creation of stable producer cell lines that synthesize virus particles carrying the MDR-internal ribosomal entry site (IRES)-lacZ and the MDR-IRES-GFP transgenes. These transcriptional fusions allow coordinate expression of Pgp and the reporter gene product to easily mark the MDR phenotype. Using the MDR-IRES-lacZ retrovirus, we demonstrate that periodic pulses of cytotoxic drug selection with a Pgp substrate enable sustained, long-term expression of the reporter beta-galactosidase in otherwise unstable transductants. We have also incorporated the improved features of GFP as a reporter gene into our MDR-IRES-GFP retrovirus. This vector allows rapid and specific identification of MDR1 gene transfer and expression in living cells either by fluorescence microscopy or by fluorescence-activated cell sorter analysis. These two MDR/reporter gene systems should be useful for in vivo studies, the evaluation of the potential of the MDR1 gene in gene therapy applications, and as a monitor of the selective efficacy of its MDR phenotype.