[Electroporation-mediated gene transduction and expression of class 1 aldehyde dehydrogenase (ALDH1) and multidrug resistance gene (MDR1)].
OBJECTIVE
This study was designed to seek an efficient and safe method for transfer of genes of large size.


METHODS
The retroviral vector containing different kinds drug resistance genes-class 1 aldehyde dehydrogenase (ALDH1) and multidrug resistance gene (MDR1) was linearized by the restriction enzyme NdeI digestion, and introduced into the packaging PA317 cells by electroporation. Selection was performed by vincristine (VCR) and 4-hydroperoxycyclophosphamide(4-HC) to obtain ALDH1 and MDR1 stably expressing cells. The integration of provirus, transcription and translation of foreign genes were confirmed by Southern blot, reverse transcription(RT)-PCR and flow cytometry, respectively. The safety of this delivery system was verified by testing helper virus(envelop gene) using nested PCR.


RESULTS
Both ALDH1 and MDR1 were successfully transduced into PA317 cells by electroporation. Stable integration of foreign genes in host cells genome was determined by Southern hybridization blot. The transcription of ALDH1 and MDR1 was demonstrated by RT-PCR. The overexpression of P-glycoprotein encoded by the downstream gene MDR1 with approximately a 4-fold increase in 98% cells was analyzed by flow cytometry. No helper virus can be detected by nested PCR assay.


CONCLUSIONS
These results implicate that the introduction and overexpression of both ALDH1 and MDR1 genes in vitro is attainable by a simple and convenient electroporation method, with the character of safety and high efficiency.
Yang
Xiao-wei
XW
Jiangsu Institute of Hematology, First Affiliated Hospital of Suzhou University, Suzhou 215006, P. R. China.
Guo
Feng
F
Wang
Wei
W
Fu
Jian-xin
JX
Cen
Jian-nong
JN
Xia
Xue-ming
XM
Chen
Zi-xing
ZX
D000444:Aldehyde Dehydrogenase / Q000235:genetics*; D000080924:Aldehyde Dehydrogenase 1 Family; D018274:Electroporation; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D019450:Genes, MDR; D015752:Genes, env; D007527:Isoenzymes / Q000235:genetics*; D050697:Retinal Dehydrogenase; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D007527:Isoenzymes; D000080924:Aldehyde Dehydrogenase 1 Family; D000444:Aldehyde Dehydrogenase; D050697:Retinal Dehydrogenase