Construction and identification of eukaryotic expressing plasmids of TGF-β type II receptor shRNA

Objective To construct eukaryotic expressing plasmid of different TGF-β type Ⅱ receptor short hairpin RNA (shRNA), inhibit their expressions in posttranscription level, identify their clones and select the clone with highest inhibition rate. Methods Four shRNA sequences synthesized by recombinant DNA technology targeting different parts of human TGF-β type Ⅱ receptor gene were cloned into eukaryotic expressing plasmid TGF-β type Ⅱ receptor shRNA expressing vector pSilencerTM 3.1-H1 neo vector shRNA1,2,3 and 4 were constructed and transfected into fibroblasts respectively by lipids. The stabilized clones inhibiting TGF-β type Ⅱ receptor expression were selected by G418. Results It was verified by partial nucleotide sequencing and restriction endonuclease digestion that the three TGF-β type Ⅱ receptor shRNA expressing vectors pSilencerTM 3.1-H1-TGF-β R Ⅱ shRNA1、2、3 were correct. pSilencerTM 3.1-H1-TGF-β R Ⅱ shRNA2 reduced mRNA peak level and protein expression dramatically by RT-PCR and Western blot Conclusion TGF-β type Ⅱ receptor shRNA expressing vectors pSilencerTM 3.1-H1-TGF-βRⅡ shRNA 1、2、3 were successfully constructed and the clone with specific and effective inhibition of TGF-β type Ⅱreceptor expression was selected.