MDR1 bicistronic vectors: analysis of selection stringency, amplified geneexpression, and vector stability in cell lines

The human multidrug resistance-1 gene (MDR1) is a dominant selectable and a mplifiable marker in mammalian tissue culture cells. MDRI is also being inv estigated as a gene therapy tool, both to protect normal cells against chem otherapy-related toxicity and to serve as an in vivo selectable marker for the overexpression of non-selectable therapeutic genes. The success of thes e strategies will depend on whether MDRI expression can be sustained at lev els high enough to confer a survival advantage on target cells. However, th e MDRI selection system is quite stringent, requiring high gene expression for transduced cells to survive in the presence of drug. The current report is a detailed molecular analysis of MDRI selection stringency compared wit h the common neo selectable marker. A bicistronic vector encoding MDRI and neo genes linked through an internal ribosome entry site was transferred in to NIH 3T3 mouse fibroblasts and K562 human leukemia cells; cells were then exposed to colchicine (to select for MDRI expression) or to G418 to select for neo expression). Surviving populations and individual clones of cells were analyzed for expression levels of MDRI and neo gene products; resistan ce to colchicine, paclitaxel, and G418; level and integrity of bicistronic mRNA; and structural integrity, integration number, and copy number of vect or DNA. These studies provide direct evidence that colchicine selection is more stringent than G418 selection; that increased selection pressure with colchicine leads to increased gene expression; that increased gene expressi on can be accommodated primarily by gene amplification, even within an indi vidual transduced clone and starting from a single-copy proviral integratio n event; and that the clonal diversity of a transduced population of cells is influenced significantly by the stringency of selection. Taken together, these results have important implications for the potential utility of MDR I as a selectable marker and as a gene therapy tool in hematopoietic cells. (C) 2001 Elsevier Science Inc. All rights reserved.