In vitro study on the cloning and transduction of human O⁶-methylguanine-DNA-methyltransferase cDNA into human umbilical cord blood CD34⁺ cells

Abstract

Objective: To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with human O⁶-methylguanine-DNA-methyltransferase (MGMT) gene could increase resistance to 1,3-Bis(2-Chloroethyl)-1-Nitrosourea (BCNU). Methods: The cDNA encoding the MGMT was isolated by using RT-PCR method from total RNA of fresh human liver, the fragment was cloned into pGEM-T vector and further subcloned into G1Na retrovirus vector. Then the GlNa-MGMT was transduced into the packaging cell lines GP+E86 and PA317 by LipofectAMINE. By using the medium containing BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, high titer amphotropic PA317 producer clone with the highest titer up to 5.8xlO⁵ CFU/ml was obtained. Cord blood CD34⁺ cells were transfected repeatedly with supernatant of retrovirus containing human MGMT-cDNA under stimulation of hemopoietic growth factors. Results: The retrovirus vector construction was verified by restriction endonuclease analysis and DNA sequencing. PCR, RT-PCR, Southern Blot, Western Blot and MTT analyses showed that MGMT drug resistance gene has been integrated into the genomic DNA of cord blood CD34⁺ cells and expressed efficiently. The transgene cord blood CD34⁺ cells conferred 4-folds stronger resistance to BCNU than untransduced cells. Conclusion: The retrovirus vector-mediated transfer of MGMT drug resistance gene into human cord blood CD34⁺ cells and its expression provided an experimental foundation for gene therapy in clinical trial.