Construction of the eukaryotic expression vector of DNMT3a shRNA and verification of its effect on silencing

Objective To construct the eukaryotic expression vector carrying double shRNA sequence targeting conserved domain of DNMT3 a mRNA and negative control( HK) in p Gensil-1 by using technology of gene recombination. Methods The recombinant plasmids were transformed into competent Escherichia coli DH5α. The positive clone E. coli was screened and enriched by kanamycin( 50 μg / ml),and then the recombinant plasmids were extracted from the DH5α and evaluated by restriction enzymes and sequence analysis. Cisplatin-resistant human lung adenocarcinoma A549 cells( A549-DDP) were chosen and transfected with the recombinant plasmids,semi-quantitative RT-PCR was performed to confirm the inhibitory rates of DNMT3 a mRNA before and after plasmid transfecting in 24,48,72 h. Results Sequence analysis showed the right sequence and the recombinant plasmids couldn't be digested by PstⅠ,and p Genesil-1-DNMT3a-shRNA transfected could significantly decrease the DNMT3 a mRNA with the inhibitory rates of 25. 5%,56. 2%,63. 4%,respectively,and the effect was dependent on the time duration. However,p Genesil-1-HK-shRNA barely affected the expression of DNMT3 a mRNA. Conclusions The eukaryotic expression vectors are successfully constructed,which laids a foundation of further research.