[Construction and assessment of short-hairpin RNA eukaryotic expression vector targeting TGF-beta1 labeled by GFP].

AIM

To construct short hairpin RNA (shRNA) eukaryotic expression vectors targeting TGF-beta1 for further research on the effects of TGF-beta1 on vasculogenesis and angiogenesis.

METHODS

Three pairs of siRNA target sequences coding from the mRNA of TGF-beta1 gene were designed and three pairs of nucleotides were synthesized. After annealing, the double-strand DNA products were ligated into the pEN_mH1c entry vector, and in turn into the shRNA eukaryotic expression vector pDS_hpEy labled by GFP through the LR recombination reaction. After sequencing successfully, the three resulting TGF-beta1 shRNA expression vectors were transfected into the mouse fibroblast cell line (NIH/3T3), and then cell clones stably expressing TGF-beta1 shRNA were screened. Reverse Transcript-Polymerase Chain Reaction (RT-PCR) and Western blot were used to detect the mRNA and protein expression.

RESULTS

RT-PCR and Western blot showed that one of the TGF-beta1 shRNA expression vectors pDS_Tc downregulated TGF-pl mRNA and protein expression markedly in NIH/3T3 cells.

CONCLUSION

ShRNA eukaryotic expression vectors targeting TGF-beta1 are successfully constructed which can be used for further investigation on the mechanism through which TGF-beta1 regulates vasculogenesis and angiogenesis.