Construction of Eukaryotic Vector Expressing shRNA of smad4 Gene

Objective 　To explore the feasibility of selective inhibiting smad4 expression using smad4 short hairpin RNA ( shRNA) interference. Methods 　Three 19bp reverse repeated motifs targeting of smad4 gene were synthesized and cloned into eukaryotic expression plasmid p Genesil-1 containing U6 shRNA promoter and termination signal of RNA polymerase. The recombinant plasmids p Genesil-shRNA1, 2, 3 and p Genesil-con were transfected into HeLa cells respectively by lipofectamine reagent . The alteration of smad4 expression was examined by RT-PCR and Western blot . Results 　It was verified by partial nucleotide sequencing and restriction endonuclease digestion that the const ructed eukaryotic vector expressing shRNA of smad4 was correct . shRNA2 in p Genesil-smad4 cells knocked down the expression of smad4 mRNA and protein dramatically compared with unt ransfected and control cells. Conclusion 　The shRNA can efficiently suppress smad4 expression in HeLa cells. The result s of the study lay the foundation for further studying on biological functions and potential application of smad4.