广西巴马小型猪小RNA启动子U6和7SK的克隆及功能验证

doi:10.3724/SP.J.1005.2012.00445

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HEREDITAS ›› 2012, Vol. 34 ›› Issue (4): 445-453.doi: 10.3724/SP.J.1005.2012.00445

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

CHEN Shi-Jin, FAN Jing, JIANG Qin-Yang, LAN Gan-Qiu, GUO Xiao-Ping, GUO Ya-Fen

College of Animal Science and Technology, Guangxi University, Nanning 530005, China

Received:2011-09-21 Revised:2012-01-14 Online:2012-04-20 Published:2012-04-25

Abstract

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.

Key words: Bama mini-pig in Guangxi, promoter, functional verification

CLC Number:

中图分类号：S8

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

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