| Diarrhea disease leads to tremendous economic losses in pig industry worldwide. Thetraditional swine diarrhea prevention and treatment methods, such as improving feedingenvironment, using antibiotics and vaccination, are becoming disappointingly less and lesseffective as ever. Therefore, an alternative and more ecological, effective method ofcontrolling this disease is urgently required. With the rapid development of modernmolecular biotechnology, to modify the characters of organisms through the transgenictechnology is becoming successful. Transgenic technology holds many advantages, such asdirectly aims at the breeding objective, time-effective, and could circumvent negativeeffects brought in by gene interactions. Most importantly, transgenic technology enable usto target multiple important economical traits at the same time, as a new and vital diseasecontrol method compared to traditional immun-methods. Hence, taking advantage oftransgenic technology to breed transgenic pigs resistant to diarrhea disease is of greatimportantance.In the present study, we described the development of two effective RNA interferencevectors, and mammary gland-specific expression vectors for antipeptides in order togenerate transgenic porcine breeding materials potentially resistant to infection of porcinediarrhoea, and the results are shown as below.1. Production of RNAi transgenic cloned pigs, targeting FUT1.①An effective RNAi combined Cre-LoxP system, which would be helpful for futuremarker free, was established, and transgenic somatic cloned blastocysts were obtained;②An effective lentivirus RNAi system was successfully developed, and seventransgenic cloned RNAi pigs were born viable. All of the transgenic pigs express EmGFPas detected by in vivo imaging. Futhermore, the disease resistance were evaluated frommany aspects;③The porcine intestinal epithelial cells (IPEC-J2) was infected by lentivirus vectorobtained in part②. Our results indicated that FUT1may be not the main pathogenic E.coli F18receptor gene;2. Mammary gland-specific expression vectors’ related studies are shown as follow.①Transgenic founder mice expressingpBD-1specifically in mammary gland wereproduced by pronuclear microinjection. It paves the way for study antimicrobia activities and its mechanism of the pBD-1in transgenic mice, and also is helpful to make transgenicmammary bioreactors in large anmimals producing pBD-1, using mouse in SPFenvironment;②A simple, efficient cell culture model for validating the structure integrity andfunctions of mammary gland-specific expression vectors was developed, representing anaccurate approach for the transgenic animals resistance breeding. The model also lay asolid basis for predicting the transgenic adult animal related immune response pathways;③Transgenic cell lines expressinghLYZ were obtained, which could support invivo development of cloned embryos produced by somatic cell nuclear transfer using thesecells as donors. Nine fetuses were havest after more than70days development in thewomb of surrogate mothers;④Transgenic cell lines and cloned embryos expressingpBD-1were obtained.Taken together and to the best of knowledge:①This is the first study reporting successfully establishment of a four promotersexpressing four shRNAs, which could eliminate all the marker genes by Cre-LoxP system,and this is the first study on FUT1gene knockdown in porcine animals.②Transgenic large animal is successfully obtained by lentivector-mediated RNAiand somatic nuclear unclear transfer.③FUT1may be not the main pathogenic E. coli F18receptor gene.④A commercial mouse mammary gland tumor cell lines can be used to validatemammary gland-specific expression vectors effectively, efficiently. Transgenic foundermice containing pBD-1is obtained, and transgenic porcine embryos expresssing pBD-1isalso support preimplantation development to blastocyst stage.The results in the present study lay a solid foundation for future creation of pigsresistant to certain diseases. |
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