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437 bp。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 PEGFP- N1 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-pU6- shEGFP 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.
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