Construction of shRNA eukaryotic expression vector of CtBP1 gene and preliminary identification of its interference efficiency

OBJECTIVE:To design and construct the eukaryotic expression vector of human CtBP1 gene short hairpin RNA(shRNA)then to transfect human endometrial carcinoma cells B-MD-C1(ADR+/+),and to investigate the silencing effect of CtBP1 shRNA on the expression of human CtBP1 gene.METHODS:siRNAs were designed by targeting human CtBP1 gene using pGenesil-1 as vector.According to the CtBP1 cDNA sequence in GenBank,2 pairs of oligonucleotides were designed.A hairpin loop with 9 base pairs was added into the interference sequence and inserted into the eukaryotic expression vectors which contain the EGFP gene,kanr gene and U6 promoter.After primer annealing,they were inserted into plasmid pGenesil-1 to construct the shRNA eukaryotic expression vector.The recombinant plasmid were transformed into E.coli DH5α strains,and the positive strains were identified by enzyme digestion and sequence analysis.The recombinant vector were transfected into B-MD-C1(ADR+/+)cells with LipofectamineTM 2000.The transfection efficiency was reported by green fluorescence protein expression and gene inhibition efficiency was analyzed by reverse transcription polymerase chain reaction(RT-PCR).RESULTS:The pGenesil-1-CtBP1 shRNA of recombinant plasmid was constructed and confirmed by DNA sequencing.Fluorescence microscope for lots of green fluorescence showed that shRNAs had transfected B-MD-C1(ADR+/+)cells(transfection rate was 60%～70%).And the CtBP1 mRNA were effectively inhibited by pGenesil-1-CtBP1-shRNA of recombinant plasmid(inhibition rate was above 50%).CONCLUSION:The eukaryoticexpression vectors of CtBP1 shRNA were constructed successfully.The shRNA could effectivelly inhibit the expression of CtBP1 gene,laiding a foundation to further study for its function and reverse the cancer multidrug resistence by blocking CtBP1 gene expression.