Construction and identification of the small hairpin RNA expression vector of Smad4/DPC4 gene

Objective:To construct and identify the small hairpin RNA(shRNA) expression vector for Smad 4/DPC 4 gene. Methods:Three shRNA sequences targeting Smad 4 were designed by software and cloned into the expression vector pGCsi-H1/Neo/GFP.DNA sequencing was used to confirm that the plasmids were constructed correctly.The constructed plasmids with different shRNA and the control plasmid were transiently transfected into the 293 cell line cells.Fluorescene quantitative PCR was used to detect the efficiency of RNA interference against Smad 4. Results:Double enzyme(HindⅢ and BamHⅠ) digestion(analysis) and DNA sequencing confirmed that Samd 4 specific shRNA expression vectors were constructed successfully.Fluorescene quantitative PCR showed that the inhibition rates of Smad 4 mRNA expression in cells transfected with shRNA expression vector psiSmad 4-1,psiSmad 4-2 and psiSmad 4-3 were 39.00%,8.80% and 73.80%,respectively,which indicated Smad 4 mRNA expression was specifically inhibited. Conclusion:Smad 4 pGCsi-H1/Neo/GFP/shRNA plasmid was constructed successfully,which may provide a novel applicable strategy for gene therapy and study of its role in carcinogenesis.