[Expression and drug resistance of human MGMT gene in hemopoietic cells mediated by bicistronic retroviral vector].
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.
Wang
J
J
Jiangsu Institute of Hematology, First Affiliated Hospital of Suzhou Medical College, Suzhou 215006, China.
Xia
X
X
Chen
Z
Z
Ruan
C G
CG
D019883:Alkyl and Aryl Transferases / Q000096:biosynthesis* / Q000235:genetics; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D002330:Carmustine / Q000494:pharmacology; D015254:DNA Modification Methylases / Q000096:biosynthesis / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D018014:Gene Transfer Techniques; D006147:Guanine / Q000096:biosynthesis; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*
D018906:Antineoplastic Agents, Alkylating; D006147:Guanine; D015254:DNA Modification Methylases; D019883:Alkyl and Aryl Transferases; D002330:Carmustine