Construction and identification of the eukaryotic vector expressing siRNA targeting human STAT3 gene

Objective:To construct siRNA eukaryotic expression vector targeting of human STAT3 gene,and identify its suppressive effect.Methods:Three specific siRNAs targeting of human STAT3 gene were synthesized and cloned into eukaryotic expression plasmid pRNAT-U6.1/neo to reconstruct recombinant plasmid pRNAT-U6.1-siRNA-STAT3,which was then identified by PCR test and DNA sequence analysis.The recombinant plasmid was transfected into human esophageal carcinoma cell line Eca-109,the positive cell clones were screened with G418,the suppression effect of STAT3 mRNA and protein was measured by RT-PCR and Western blot to select the optimal siRNA.Results:PCR test and DNA sequence analysis showed that the recombinant plasmid was constructed successfully.RT-PCR and Western blot analyses demonstrated that the optimal siRNA which could effectively silence the target gene pRNAT-U6.1-siRNA3.Conclusion:The siRNA eukaryotic expression vector targeting of human STAT3 gene was constructed successfully,which could inhibit the expression of STAT3 gene.