Production Of Transgenic Porcine Breeding Materials Potentially Resistant To Porcine Diarrhoea

Piglet diarrhea is one of the most serious diseases in pig industry,leading to tremendous economic losses worldwide at present.The virus and E.coli are the most important pathogenic agents.Transmissible gastroenteritis virus(TGEV)and porcine epidemic diarrhea virus(PEDV)are the main pathogens responsible for piglet viral diarrhea.K88 and F18 are the main pathogens responsible for piglet bacterial diarrhea.Hence,breed new pig variety which is resistant to diarrhea disease is of great importantance for pig industry.Knock-out of the pathogenic host cellular receptor and induction resistance gene may represent an effective strategy to investigate the gene function and accelerate the livestock resistant breeding progress.In the present study,we described the development of gene site-specific edited,multiple promoter and shRNAs RNA interference,mammary gland-specific multiple genes co-expression technology in order to generate transgenic porcine breeding materials potentially resistant to infection of piglet diarrhoea,and the results are shown as below:1)We site-specific edited the target pAPN which encode a potential receptor for TGEV、PEDV and K88,using TALEN and homologous recombination techonology,Site-specific editing of pAPN reduced TGEV proliferation in ST cells by 96～99%at different time periods post-infection and typical cytopathic effects were delayed.Next,five pAPN site-specific clone pigs were successfully obtained.we report the proteomic analysis of pAPN site-specific edited、TGEV-infected and control ST cells using TMT labeling quantitative proteomic and bioinformatic analysis.The DPP4 and SLA-1 were up-regulated in the pAPN site-specific edited cells,we inferred that DPP4 and SLA-1 were important for TGEV infection.TGEV infection induced ST cell cycle arrest,cell apoptosis and active the natural immune pathway.We found that IFN-α、IFN-β、IFN-γand ISG15 can inhibit the proliferation of TGEV in ST cells.Furthermore,the positive and negative selection site-specific knock-in targeting pAPN containing IFN-α、FN-β、IFN-γ、ISG15、IFN-αβγ vectors were constructed.2)Four promoters and four shRNAs vector with LoxP sites at each end of the selectable marker genes that target PEDV and TGEV was constructed,then porcine fetal fibroblasts was obtained after G418 selection.Finally,we successfully obtained transgenic SCNT porcine blastocysts.3)A mammary gland bioreactor co-vector whicih carried six anti-bacterial and/or anti-viral genes was constructed,then porcine fetal fibroblasts was obtained after G418 selection.Finally,we successfully obtained transgenic SCNT porcine blastocysts.