[Efficient expression of multidrug resistance gene mdr1 in retroviral vector under control of an internal ribosome entry site].
OBJECTIVE
To test the efficiency of human mdr1 gene expression under the control of an internal ribosome entry site (IRES).


METHODS
The expression retroviral vector Halmdr1 was constructed from pSXLC/pHa system which contains an IRES from encephalomyocarditis virus. The vector was introduced into ecotropic packaging cells GP + E86 by liposome-mediated transfection. The retrovirus producing cells were obtained by 50 micrograms/L vincristine selection. The success of transfer and expression of mdr1 gene were determined by polymerase chain reaction (PCR) and flow cytometry (FCM).


RESULTS
Virus in the supernatant of the producer cells, in which the integration of mdr1 gene was confirmed by PCR, was titrated to 2.0 x 10(5) cfu/ml. The selected producer cells exhibited a 24-52-fold increase of resistance to vincristine, daunorubicin and taxol in comparison with control cells GP + E86. The expression of mdr1 gene was confirmed by both reverse transcription PCR at RNA level and FCM at protein level, although the HaImdr1 transfectants showed somewhat less expression of P-gp when compared to that with cap-dependent translation of Ha mdr1.


CONCLUSIONS
mdr1 gene can be effectively translated and expressed under the control of IRES in cap-independent manner, it may be used as a dominant selectable marker in bicistronic vectors for gene therapy.
Jiangsu Institute of Hematology, First Affiliated Hospital of Suzhou Medical College, Suzhou 215006.
J
J
Fu
Z
Z
Chen
W
W
Wang
D000818:Animals; D003630:Daunorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D004686:Encephalomyocarditis virus / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D019450:Genes, MDR; D005822:Genetic Vectors; D006023:Glycoproteins / Q000096:biosynthesis*; D006801:Humans; D051379:Mice; D017239:Paclitaxel / Q000494:pharmacology; D012190:Retroviridae / Q000235:genetics; D012270:Ribosomes / Q000235:genetics; D014162:Transfection; D014750:Vincristine / Q000494:pharmacology
D006023:Glycoproteins; D014750:Vincristine; D017239:Paclitaxel; D003630:Daunorubicin