Comparison of methotrexate resistance conferred by a mutated dihydrofolatereductase (DHFR) cDNA in two different retroviral vectors

We previously reported the protection of hematopoietic cells from methotrex ate (MTX) toxicity using an N2-based double copy vector containing serine 3 1 (S31)-mutated dihydrofolate reductase (DHFR) (DC/SV6S31). To examine whet her the use of SFC-bascd dicistronic vectors will lead to improvement in ge ne transfer over the DC/SV6 vector, we compared the protection provided by MTX to NIH3T3 cells and hematopoietic progenitor cells infected with these retroviral constructs containing the S31 variant DHFR cDNA. In NIH3T3 cells , the 50% effective dose values of MTX conferred by the SFG vector were 8-f old higher than those obtained with the DC/SV6 vector. DHFR mRNA levels wer e 22-fold and 38-fold higher than that seen for the DC/SV6 vector according to Northern blot and real-time polymerase chain reaction analysis, respect ively. However, DHFR protein expression and DHFR enzyme activity were only 1.5-fold and 2-fold higher in the SFG vector, respectively, indicating that the mRNA from the SFG vector is translated less efficiently than the mRNA generated from the DC/SV6 vector. Furthermore, the degree of MTX protection conferred by each vector in both mouse and human hematopoietic cells was t he same. These results indicate that the in vitro transduction efficiency a nd transgene expression of human DHFR in hematopoietic progenitor cells is equally conferred by both vectors.