Bicistronic retroviral vectors for combining myeloprotection with cell-surface marking

We have developed a retroviral vector coexpressing the multidrug-resistance 1 (MDR1) cDNA for inducing cancer drug resistance and the truncated versio n of the few-affinity nerve growth factor receptor (Delta LNGFR) for cell-s urface marking of transduced cells. The vector is based on the FMEV backbon e which mediates high levels of gene expression in hematopoietic: cells. To achieve optimal expression levels of both cDNAs, untranslated regions from MDR1 and Delta LNGFR were removed and three different connections were tes ted: retroviral splice signals, an internal ribosomal entry site (IRES) fro m encephalomy-ocarditis virus, and an internal promoter from the chicken be ta-actin gene. As determined by two-color flow cytometry, the best correlat ion of the expression of both cDNAs was 1 obtained using the vector SF1mS d elta which utilized retroviral splice signals for co-expression. Simultaneo us expression of both cDNAs at the single cell level was also shown by conf ocal laser microscopy. Lymphoid and hematopoietic, progenitor cells, includ ing primary human CD34(+) cells, transduced with SF1mS delta acquired domin ant multidrug resistance. Transduced primary CD34(+) cells could be enriche d in vitro based on expression of Delta LNGFR, avoiding exposure to cytosta tic agents. Thus, monitoring the selection of chemotherapy-resistant cells and analyzing their biological properties may be alleviated both in vitro a nd in vivo.