Characterization and comparison of porcine H1, chicken H1 and mouse H1 RNA polymerase Ⅲ promoter for short hairpin RNA expression
Object: U6 and 7SK RNA polymerase Ⅲ promoters are widely used to express short-hairpin RNA (shRNA)in RNA interference(RNAi) research.However,their toxicity has caused concern;thus, a relatively weak H1 promoter may be optimal.Method:Here,porcine genome DNA was used as a template for porcine H1 PCR amplification, which replaced the cytomegalovirus and T7 promoter of eukaryotic expression vector pcDNA3.1 (+).A designed siRNA sequence for the EGFP gene was inserted into pcDNA3.1 (+)to produce siRNA specifically targeting EGFP(ppH1-shEGFP).The same was performed for mouse and chicken H1 promoter interference vectors, pmH1-shEGFP and pchH1 -shEGFP.PK and DF-1 cells were transfected with interference expression vectors.The mean fluorescence intensity of EGFP was measured by fluorescence microscopy and flow cytometry.In addition,EGFP and shRNA expression was measured by quantitative PCR.Result:Sequencing confirmed the core sequence of porcine,chicken and mouse H1 contained typical elements of RNA polymerase Ⅲ type promoter.Expressed shRNAs for the porcine H1 promoter resulted in EGFP -specific knockdown at lower levels than chicken and mouse H1.Conclusion: Thus,porcine RNA polymerase Ⅲ type H1 promoter may be useful for knockdown gene expression to study functional genomics and for the development of porcine RNAi technology and transgenic porcine research.
H1 RNA polymerase Ⅲ promoter short hairpin RNA expression characterization comparison
Qing-Wen Meng Hai-Chang Yin Wei Wang Hong-Yan Chen
National Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences,150001
国内会议
沈阳
英文
286-291
2015-08-01（万方平台首次上网日期，不代表论文的发表时间）