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Yi Chuan ; 34(4): 445-53, 2012 Apr.
Article in Chinese | MEDLINE | ID: mdl-22522162

ABSTRACT

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.


Subject(s)
Promoter Regions, Genetic/physiology , RNA Polymerase III/genetics , RNA, Small Interfering/genetics , Swine, Miniature/genetics , Animals , Base Sequence , Cloning, Molecular , Molecular Sequence Data , Sequence Analysis, DNA , Swine
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