title: [In vitro study on transduction of human O(6)-methylguanine-DNA-methyltransferase cDNA into human umbilical cord blood CD34(+) cells].
abstract: 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.
authors: The First Affiliated Hospital of Suzhou Medical College, Jiangsu Institute of Hematology, Suzhou, Jiangsu, 215006 P.R.China. jswang@shmu.edu.cn
authors: J
authors: J
authors: Wang
authors: X
authors: X
authors: Xia
authors: Z
authors: Z
authors: Chen
authors: D
authors: D
authors: Lu
authors: J
authors: J
authors: Xue
authors: C
authors: C
authors: Ruan
mesh_terms: 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
chemical_list: D018952:Antigens, CD34; D018076:DNA, Complementary; D019853:O(6)-Methylguanine-DNA Methyltransferase