Construction and expression of recombinant retroviral vectors expressing siRNA for MDR1 gene / 吉林大学学报(医学版)

ABSTRACT

Objective To construct and identify the recombinant retroviral vectors expressing siRNA for MDR1 gene knock-down,sequentially to detect the reversing effect of RNAi on the multidrug resistant phenotype in MCF-7/ADR cells. Methods The target sequences of siRNAs for MDR1 knock-down were designed by IDTdna online. The DNA sequences containing a palindrome structure were synthesized and annealed to form double-stands which were inserted into linear pSUPER vectors later. The recombinant vectors were identified by agarose gel electrophoresis after cleaving of double enzymes and by sequencing,then were transfected into 293T packaging cells which produced retroviral particles in supernatant,the retrovirus soup was used to infect MCF-7/ADR cells. The expression level of MDR1 was detected from both mRNA level and protein level by RT-PCR and Western blotting,respectively. Results The recombinant pSUPER vectors expressing siRNA were confirmed by agarose gel electrophoresis after cleaving of double enzymes and by sequence analysis. The inserted oligonucleatide sequences were identical to the original sequences and in corresponding position. Compared with control group,the inhibitory effects of pSUPER.retro-MDR1 and pSUPER.retro-MDR2 were significant. Conclusion pSUPER recombinant retroviral vectors expressing siRNA for MDR1 knock-down is construted successfully,and it has inhibitory effect on the expression of MDR1 gene in MCF-7/ADR cells.