广西巴马小型猪小RNA启动子U6和7SK的克隆及功能验证

Cloning and functional verification of U6 and 7SK promoter of small RNA from Bama mini-pig in Guangxi

为了探讨巴马小型猪启动子U6和7SK的功能以及为生产GGTA1基因沉默的广西巴马小型猪奠定基础,文章克隆并分析了siRNA启动子U6和7SK,并分别连接pMD18-shEGFP载体,分别与PEGFP-N1共转猪肾细胞,进行RNAi实验。以pMD18-hU6-shEGFP为阳性对照,无启动子的pMD18-shEGFP载体为阴性对照,单独转染PEGFP-N1为第一组空白对照,以ddH2O替代质粒为第二组空白对照组,验证这两种启动子在猪细胞中的功能。结果表明:广西巴马小型猪RNA聚合酶III型siRNA启动子U6和7SK的序列全长分别为553 bp和437bp。成功构建了pMD18-pU6-shEGFP和pMD18-p7SK-shEGFP干扰载体,转染猪源PK-15细胞,证明U6以及7SK两个启动子具有较高的siRNA表达活性,可以用于α-1,3半乳糖转移酶等猪源基因的沉默实验。

To investigate the functions of U6 and 7SK of Bama mini-pig and produce Bama mini-pig with silenced GGTA1 gene,the siRNA promoters U6 and 7SK were cloned,ligated into pMD18-shEGFP,and co-transfected with PEGFPN1 into PK-15 kidney cells of pigs to be used in RNAi experiments.The functions of the two promoters in pig cells were verified using pMD18-hU6-shEGFP as the positive control,pMD18-shEGFP vector without promoter as the negative control,PEGFP-N1 as the first blank control,ddH2O in replacement of the plasmid as the second blank control.The results showed that the lengths of U6 and 7SK in Bama mini-pig were 553 bp and 437 bp,respectively.Vectors pMD18-pU6shEGFP and pMD18-p7SK-shEGFP were constructed and transfected into PK-15 cells from pigs.Promoters pU6 and p7SK proved to express high levels of siRNA activity and can be used in the experiment of silencing α-1,3galactosyltransferase gene.