Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (1): 170-178.doi: 10.3864/j.issn.0578-1752.2013.01.020

• VETERINARY SCIENCE • Previous Articles     Next Articles

Establishment of Cell Lines Transcribing shRNA Targeted to Jiv Gene and Constuction of CSFV Resistant Transgenic Piglet

 GUO  Kang-Kang, LEI  An-Min, NING  Peng-Bo, CHENG  Min, HE  Lei, LIU  Wei, TAN  Xue-Chao, XU  Lei, CAO  Wei-Wei, ZHANG  Yan-Ming   

  1. College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi
  • Received:2012-11-02 Online:2013-01-01 Published:2012-11-15

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Abstract: 【Objective】To construct postive cell strans inserted the shRNA fragments targeting to porcine J-Domain protein interacting with virus gene, the interference effects were evaluated by comparing the proliferation of classical swine fever virus (CSFV) in constructed cell strains and the cell strain with significant CSFV resistance would be provided as the basic materials for constructing the CSFV-resistant transgenic pig. 【Method】 Four shRNA interfering fragments were designed tagerting to pig Jiv gene and 4 lentivirus inserted the designed shRNA fragment（named P1, P2, P3 and P4）. Four positive PK-15 cell strains transfected with lentivirus were established individually and the interference effects of CSFV were evaluated by determining the RNA of CSFV in positive cell strains after 72 h infected with CSFV by real-time RT-PCR . The porcine fetus fibroblasts were isolated and infected with lentivirus P2 which interfered the proliferation of CSFV remarkerably in cell. A positive porcine fetus fibroblast strain stable expressing the shRNA interfering fragment targeting to Jiv gene was established which also was the donor cells for constructing the transgenic pig. The nuclear of positive pircine fetus fibroblast was transferred into matured enucleated porcine oocytes, and somatic embryos were obtained and transplanted into the receptor sows for constructing transgenic pig. The foreign gene was identified in obtained transgenic pig.【Result】 Four PK-15 cell strains inserted into the shRNA interfering fragments targeting to the Jiv gene of porcine were established, the P2 cell strain (inserted into P2 interfering fragment) was significantly CSFV-resistant. The nuclear of porcine fetus fibrlblast inserted P2 interfering fragment was donor cells for constructing transgenic pig by somatic cell cloning and embryo transplantation, and a transgenic piglet was obtained.【Conclusion】 The expression of Jiv gene can influence the proliferation of CSFV in cells. A PK-15 cell strain with stable expressing shRNA P2 was established which could resist CSFV proliferation remarkablly. A trnasgenic piglet was constructed by somatic cell cloning and embryo transplantation which carries the P2 interfering fragment targeting to porcine Jiv gene.

Key words: classical swine fever virus , RNA interference , lentivirus , transgenic pig

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