IN VITRO STUDY ON THE CLONING AND TRANSDUCTION HUMAN O6-METHYLGUANINE-DNA-METHYLTRANSFERASE cDNA INTO HUMAN UMBILICAL CORD BLOOD CD34+ CELLS OF
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)-l-Nitrosourea (BCNU).
Methods: The eDNA encoding the MGMT was isolated by using RTPCR method from total RNA of fresh human liver, the fragment was cloned into pGEM-T vector and further subcloned into GINa retrovirus vector. Then the G1Na- 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 5.8×105 CFU/ml was obtained. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human MGMTcDNA 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.
Methods: The eDNA encoding the MGMT was isolated by using RTPCR method from total RNA of fresh human liver, the fragment was cloned into pGEM-T vector and further subcloned into GINa retrovirus vector. Then the G1Na- 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 5.8×105 CFU/ml was obtained. Cord blood CD34+ cells were transfected repeatedly with supernatant of retrovirus containing human MGMTcDNA 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.