[In vitro study on transduction of human O(6)-methylguanine-DNA-methyltransferase cDNA into human umbilical cord blood CD34(+) cells].
OBJECTIVE
To explore human umbilical cord blood hematopoietic progenitor cells transduced with human O(6)-methylguanine-DNA-methyltransferase (MGMT) gene increase resistance to 1,3-Bis(2-Chloroethyl)-1-Nitrosourea(BCNU).


METHODS
The present authors obtained a full length cDNA fragment encoding the human MGMT from a patient with cholelithiasis liver tissue by RT-PCR method and confirmed by DNA sequencing. The fragment was cloned into pGEM-T vector and further subcloned into G1Na retrovirus vector. Then the G1Na-MGMT was transfected into the packaging cell lines GP+E86 and PA317 by LipofectAMINE method; using the medium containing BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, the authors obtained high titer amphotropic PA317 producer clone with the highest titer up to 1.6x10(6) CFU/ml. Cord blood CD34(+) cell were transfected repeatedly with supernatant of retrovirus containing human MGMT cDNA under stimulation of hemopoietic growth factors.


RESULTS
PCR, RT-PCR, Southern blot, Northern blot, Western blot and MTT analyses showed that MGMT gene had been integrated into the genomic DNA of cord blood CD34(+) cells and expressed efficiently in the transfected cells. The transgene recipient cells conferred 4 folds stronger resistance to BCNU than that of the non-transduced.


CONCLUSIONS
The retrovirus vector-mediated transfer of MGMT drug resistance gene into human cord blood CD34(+) cells and expression could confer the resistance of transgene cells to BCNU toxicity.
Wang
J
J
The First Affiliated Hospital of Suzhou Medical College, Jiangsu Institute of Hematology, Suzhou, Jiangsu, 215006 P.R.China. jswang@shmu.edu.cn
Xia
X
X
Chen
Z
Z
Lu
D
D
Xue
J
J
Ruan
C
C
D018952:Antigens, CD34 / Q000032:analysis*; D018076:DNA, Complementary / Q000235:genetics*; D005312:Fetal Blood / Q000378:metabolism*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics
D018952:Antigens, CD34; D018076:DNA, Complementary; D019853:O(6)-Methylguanine-DNA Methyltransferase