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

Yi Chuan. 2012 Apr;34(4):445-53. doi: 10.3724/sp.j.1005.2012.00445.
[Article in Chinese]

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Cloning, Molecular
  • Molecular Sequence Data
  • Promoter Regions, Genetic / physiology*
  • RNA Polymerase III / genetics*
  • RNA, Small Interfering / genetics*
  • Sequence Analysis, DNA
  • Swine
  • Swine, Miniature / genetics*

Substances

  • RNA, Small Interfering
  • RNA Polymerase III