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 pGenesil-1 containing U6 shRNA promoter and termination signal of RNA polymerase. The recombinant plasmids pGenesil-shRNA1, 2, 3 and pGenesil-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 constructed eukaryotic vector expressing shRNA of smad4 was correct. shRNA2 in pGenesil-smad4 cells knocked down the expression of smad4 mRNA and protein dramatically compared with untransfected and control cells. Conclusion The shRNA can efficiently suppress smad4 expression in HeLa cells. The results of the study lay the foundation for further studying on biological functions and potential application of smad4.