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

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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.

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Acknowledgments

We are indebted to professor Zhengxing Lian for providing 3s-loxP-GFP-TLR4, we thanks Mr. Qi Wang and Hui Feng for providing experimental supplies. This work was supported by the National Transgenic Breeding Program of China (2009ZX08006-004B).

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Authors and Affiliations

  1. Anhui Provincial Laboratory of Local Livestock and Poultry Genetical Resource Conservation and Breeding, College of Animal Science and Technology, Anhui Agricultural University, No.130 of Changjiang West Road, Hefei, 230036, Anhui, China

    Jian-wen Chen, Yu Zhang, Yuan-liang Zhang, Chao Wei, Xing Liu, Na-ru Zhou, Qing Jia, Yun-sheng Li, Xiao-rong Zhang & Yun-hai Zhang

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  1. Jian-wen Chen
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  2. Yu Zhang
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  3. Yuan-liang Zhang
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  4. Chao Wei
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  5. Xing Liu
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  6. Na-ru Zhou
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  7. Qing Jia
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  8. Yun-sheng Li
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  10. Yun-hai Zhang
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Correspondence to Yun-hai Zhang.

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Chen, Jw., Zhang, Y., Zhang, Yl. et al. Construction of multiple shRNAs expression vector that inhibits FUT1 gene expression and production of the transgenic SCNT embryos in vitro. Mol Biol Rep 40, 2243–2252 (2013). https://doi.org/10.1007/s11033-012-2287-3

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  • DOI: https://doi.org/10.1007/s11033-012-2287-3

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