High-Efficiency Retroviral Vector Containing Human Mutated Dihydrofolate Reductase cDNA and Its Expression in Murine Hematopoietic Progenitor Cells / 中国肿瘤生物治疗杂志

ABSTRACT

A double-copy Moloney leukemia virus-based retroviral vector containing both the Neo~(R) gene and a mutant human dihydrofolate re-ductase(S31 mutation) cDNA was packaged into the Amphotropic packaging cell hne GP-EAM12( AM12), and a Amphotropic producer cell hne (named AM12-S31)was obtained. In this study, we investigated its drug resistant characteristics, viral titer and for murine hematopoietic progenitor cells transduction as well. MTT assay verified that the AM12-S31 cells were resistant to G418 and methotrexate(MTX), the IC50 were more than 800 ?g/ml and 100 ?M respectively while the control cell line AM12 was sensitive to both drugs, the IC50 were 180 ?g/ml and 10 ?M, respectively. The viral titer for this cell line was approximately 7.8? 104~4.2? 105 G418-resistant colony forming units/ml. The replication-competent virus can not be detected in this producer cell line. We also use the AM12-S31 cells to transfect murine hematopoietic cells (By coculture) . The positive colonies were found in all the G418 concentrations using CFU-GM assay. No G418-resistant colony was found using AM12 transfection. The infected murine marrow cells were returned to lethally irradiated(900rad)recipients. The murine transplanted with AM12-S31 infected marrow cells showed protection from lethal MTX toxicity as compared with AM12 infected animals. Evidence for integration and the proviral DNA was obtained by PCR amplification of proviral DNA. These results indicated this producer cell hne could produce high titer, high-efficiency and non-replcational competent virus. The murine marrow cells could be transfected successfully using this system, and express the foreign gene. The lethal irradiated murine marrow function could be reinstitution by infusing the hematopoietic progenitor cells tranducted with human mutant dihydrofolate reductase. In my opinion, this system would play an important role in research the long-term protection of murine marrow hematopoietic function and drug resistant gene therapy.