Objective: To increase myeloid progenitors resistance to chemotherapy and prevent myelosuppression caused by alkylating agents.
Methods: Total cellular RNA was extracted from human liver and cDNA was synthesized by superscript reverse transcriptase, a polymerase chain reaction(PCR) was conducted. We obtained a full length cDNA fragment encoding human alkyguarine-DNA-alhyltransferase(MGMT). The PCR product was cloned into pGEMT-T vector and further subcloned into G1Na retrovirus expression vector. Then the recombinant plasmid was transduced into the packaging cell lines GP+E86 and PA317 by lipofect AMINE.
Results: By using the medium containing BCNU for cloning selection and ping-ponging supernatant infection between ecotropic produced clone and amphotropic producer clone, we obtained high titer amphotropic PA317 producer clone with the highest titer up to 8.6-10 CFU/ml. Human hematopoietic cells were infected repeatedly with this high titer virus under stimulation of hemopoietic growth factors IL-3, IL-6 and SCF. PCR, RT-PCR, Southern blot, Western blot and MTT analyses showed that MGMT gene has integrated into the genomic DNA of human hemopoietic cells and expressed efficiently.
Conclusions: This study provides a foundation for application of gene therapy to tumor clinical trial.