[Construction of eukaryotic expression vector of short hairpin RNA for transforming growth factor-beta1]
Jing Wang1, Jun-Zheng Wu, Fu-Ping Guo
1Dept. of Biology, College of Stomatology, The Fourth Military Medical University, Xi'an 710032, China.
Objective:
To construct the plasmid containing short hairpin RNA (shRNA) of TGF-beta1 expression vector.
Methods:
Short chain oligonucleotide was designed according to the TGF-beta1 mRNA sequence provided by Genebank, then DNA segment was gained through annealing after chemosynthesis, and then was cloned to pWH1 vector. The recombinant TGF-beta1 shRNA expression vector was evaluated by using enzyme cutting. At last, the constructed TGF-beta1 expression vector was transfected into salivary gland mucoepidermoid carcinoma (Ms) cells by Lipofectomine TM 2000, and its effect on TGF-beta1 expression was observed by RT-PCR and immunohistochemistry.
Results:
Successful construction was identified by enzyme cutting and the constructed plasmid was called pWH1-TGF-beta1. The shRNA and it inhibited the TGF-beta1 mRNA and protein expression effectively.
Conclusion:
The constructed TGF-beta1 shRNA expression vector can block the TGF-beta1 expression in salivary gland mucoepidermoid carcinoma cells.
