NDLI: Construction of multiple shRNAs expression vector that inhibits FUT1 gene expression and production of the transgenic SCNT embryos in vitro

Content Provider	Springer Nature Link
Author	Chen, Jian wen Zhang, Yu Zhang, Yuan liang Wei, Chao Liu, Xing Zhou, Na ru Jia, Qing Li, Yun sheng Zhang, Xiao rong Zhang, Yun hai
Copyright Year	2012
Abstract	Enterotoxigenic Escherichia coli F18 is a major pathogen that causes postweaning diarrhoea and edema disease in piglets. The alpha(1,2)-fucosyltransferase (FUT1) gene has been identified as an ideal candidate gene for controlling the expression of the receptor for ECF18 bacteria. Therefore, the use of RNA interference (RNAi) to study the function of the FUT1 gene and to produce FUT1 knockdown transgenic pig would be highly beneficial. We developed an effective strategy for the expression of multiple small hairpin RNA simultaneously using multiple RNA polymerase III (hU6, hH1, mU6 and h7SK) promoters in a single vector to knockdown the FUT1 gene. Stable FUT1 knockdown transgenic fibroblast lines were generated by transfecting porcine fetal fibroblasts with the constructed vectors. Real-time RT-PCR indicated that the mRNA level of FUT1 in the transgenic fibroblast lines was significantly lower than that in the control, as much as 29 %. Finally, we successfully obtained transgenic SCNT porcine embryos. Overall, the results demonstrated that this vector-based RNAi expression system is an efficient approach to knockdown FUT1 gene expression in porcine fetal fibroblast cells, which could thereby provide donor cells for somatic cell nuclear cloning and the potential production of a marker-free transgenic pig resistant to F18 related diseases. Furthermore, it also provides strong evidence that this approach could be useful both in the production of transgenic livestock resistant to disease, and in the development of effective strategies for the suppression of gene expression in clinical gene therapy.
Starting Page	2243
Ending Page	2252
Page Count	10
File Format	PDF
ISSN	03014851
Journal	Molecular Biology Reports
Volume Number	40
Issue Number	3
e-ISSN	15734978
Language	English
Publisher	Springer Netherlands
Publisher Date	2012-12-01
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Escherichia coli F18 FUT1 RNAi Marker-free Multiple shRNAs Animal Anatomy / Morphology / Histology Animal Biochemistry
Content Type	Text
Resource Type	Article
Subject	Genetics Medicine Molecular Biology

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