[A retroviral bicistronic strategy for efficient coexpression of ALDH1 and mdr1].
OBJECTIVE
To elucidate the feasibility of coexpression of human class 1 aldehyde dehydrogenase (ALDH1) and multidrug resistance gene (mdrl) by a retroviral bicistronic vector containing an internal ribosome entry site (IRES).


METHODS
The constructed retroviral bicistronic vector G1Na-ALDH1-IRES-mdr1 (G1Na-AIM) was transduced into amphotropic packaging cells PA317 by electroporation. Selection was performed by using 15 ng/ ml vincristine. The titer of retrovirus was elevated by ping-pong transduction. K562 cells were transfected with retroviral producing cell supernatant. The integration of the transferred gene and its expression and the efficiency of transfection were determined by polymerase chain reaction (PCR), Southern blot, MTT assay, flow cytometry (FCM) and colony forming assay, respectively.


RESULTS
The retroviral titer reached 1.0 x 10(5) cfu/ml in the supernatant of the PA317/ AIM cells after cross-infection with ecotropic GP + E86 cells. The transfected K562 cells exhibited a 3 - 10-fold increase of resistance to 4-hydroperoxycyclophosphamide (4-HC) and vincristin (VCR) and 62% of them expressed P-gp. The integration of both ALDH1 and mdr1 was confirmed by PCR and Southern blot analysis.


CONCLUSIONS
The IRES containing bicistronic retroviral vector allows functional coexpression of two different kinds of drug resistance genes. This strategy could be applicable to the transfer of other combinations of drug resistance gene and dominant selection of transfectant cells in vivo.
Yang
X
X
Jiangsu Institute of Hematology, First Affiliated Hospital of Suzhou University, Suzhou, China.
Chen
Z
Z
Wang
W
W
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D000444:Aldehyde Dehydrogenase / Q000235:genetics*; D000080924:Aldehyde Dehydrogenase 1 Family; D005822:Genetic Vectors; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D020014:K562 Cells; D050697:Retinal Dehydrogenase; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection / Q000379:methods*
C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D007527:Isoenzymes; D000080924:Aldehyde Dehydrogenase 1 Family; D000444:Aldehyde Dehydrogenase; C510223:ALDH1A1 protein, human; D050697:Retinal Dehydrogenase