Construction of the eukaryotic expression vector carrying double shRNA sequence targeting the conserved domain of gene DNMT3b

Objective To construct the eukaryotic expression vector carrying double shRNA sequence targeting conserved domain of DNMT3b and identify the nucleic acid sequence.Methods To design three strands shRNA targeting conserved domain of DNMT3b mRNA and a negative control(HK) were designed,and cloned into eukaryotic expression vectors pGensil-1 by using technology of gene recombination,and named P-DNMT3b-shRNA1,P-DNMT3b-shRNA2,P-DNMT3b-shRNA3 and P-HK.The constructed recombinant plasmids were transfected into E.coli DH5α,and inoculated into LB flat plates.Five monoclonal colonies were amplifisised,and extracted with Kana.The sequence of the plasmids were evaluated by electrophoresis and sequence analysis.Results Two gene-fragments were isolated from the recombinant plasmids by Sal Ⅰ,with the smaller one being 440 bp,and the larger one being 4.55 kb respectively.At the same time,the cloning gene was detected by sequence analysis,and the results were completely correct.Conclusion The eukaryotic expression vectors are successfully constructed.