[Study on expression and resistance of the double drug resistance genes transduced into human umbilical cord blood CD34+ cells mediated by bicistronic retroviral vector].
To explore whether human umbilical cord blood CD34+ cells transduced with human aldehyde dehydrogenase class-1 (ALDH1) and multidrug resistance gene (MDR1) increase resistance to 4-Hyaroxycyclophosphophamide (4-HC) and P-Glycoprotein Effluxed Drugs, a bicistronic Retroviral vector G1Na-ALDH1-IRES-MDR1 was constructed. The vector was transduced into the packaging cell lines GP + E86 and PA317 by LipofectAMINE. Using the medium containing VCR and 4-HC for cloning selection and pingponging supernatant infection between ecotropic producer clone and amphotropic producer clone, we obtained high titer amphotropic PA317 producer clone with the highest titer up to 5.6 x 10(5) CFU/ml. Cord blood CD34+ cells were transfectced repeatedly with supernatant of retrovirus containing human ALDH1 and MDR1cDNA under stimulation of hemopoietie growth factors. PCR, RT-PCR, Southern blot, Northern blot, FACS and MTT method analyses show that dual drug resistance genes have been integrated into the genomic DNA of cord blood CD34+ cells and expressed efficiently. The transgenes recipient cells confered 4- to 7.2-folds stronger resistance to cyclophospsphamede and P-Glycoprotein Effluxes drug in comparison with the nontransduced cells. This study provided a foundation for the application of combination chemotherapy in tumor clinical trial.
Wang
J S
JS
First Affiliated Hospital, Suzhou Medical College, Jiangsu Institute of Hematology, Suzhou 215006.
Xia
X M
XM
Chan
Z X
ZX
Lu
D R
DR
Xue
J L
JL
Ruan
C G
CG
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000235:genetics; D000444:Aldehyde Dehydrogenase / Q000235:genetics*; D000080924:Aldehyde Dehydrogenase 1 Family; D018952:Antigens, CD34 / Q000032:analysis*; D002478:Cells, Cultured; D005312:Fetal Blood / Q000166:cytology*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D019450:Genes, MDR; D015316:Genetic Therapy; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D050697:Retinal Dehydrogenase; D012190:Retroviridae / Q000235:genetics*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018952:Antigens, CD34; D007527:Isoenzymes; D000080924:Aldehyde Dehydrogenase 1 Family; D000444:Aldehyde Dehydrogenase; C510223:ALDH1A1 protein, human; D050697:Retinal Dehydrogenase