Preliminary Study On Generation Of Induced Pluripotent Stem Cell Lines From Tibet Minipig

Induced pluripotent stem cells are constantly self-renewing and multipotential cells acquired from somatocyte reprogramming induced by expressing some specific transcription factors in differentiated cells. IPS cells, similar to embryonic stem cells in cell shape, growth characteristics, surface markers and teratoma formation, have advantages of avoiding immunoreactions and not involving moral problems, so that they have extensive and important clinical application value. IPS cells are used to cultivate embryonic stem cells of animal models or important economic animals, which are failed to be acquired from animal embryos through traditional methods. In this way, the cells can be genetically modified and inheritance and breeding can be done.The iPS cell line of mouse and rat has been established, and it has been used in the research of the disease model of human. However, there are large differences between mouse and human in figure, physiological characteristics and lifespan. Comparatively, pigs are more approximate to human in dissection, physiological functions, biochemical and metabolic processes and etc. Therefore, pig iPS cell line establishment is more favorable towards the research of human disease mechanism and the establishment of effective therapeutic model. In recent years, it is an increasingly common trend to use pigs as medical experimental animals, and especially the establishment of various kinds of miniature porcine strains promotes the development and application in this field. Moreover, the application of transgene and clone technology increases that of using pigs as model of human disease. There would be broad prospects in using transgenic technology to make transgenic porcine animal model of human disease taking miniature pigs as material. In 2004, Professor Gu Wei-wang, from the Center of Experimental Animals in Southern Medical University, introduced Tibet pigs from Tibet Autonomous Region into Guangzhou with others. Through five years of acclimatization, they cultivated a new kind of miniature pigs, named "Tibet minipig". At the same time, they were conducting a comparatively systematic research on the Tibet minipig, in biological characteristic aspects of the physiological-biochemical characteristics, productive performance, genetic polymorphisms and etc. The Tibetan miniature pig is a national recognized experimental kind of miniature pig.According to this, this experiment plans to:1) establish technology of lentivirus-mediated in vitro delivery system of foreign genes based on PEFs, in order to provide technical support for the next step, that is to deliver Oct4, Sox2, Klf4 and c-Myc into the fibroblast of the Tibet minipig, with the help of the lentiviral vector method; 2) produce high-effectively PEFs infected virus vectors, carrying Oct4, Sox2, Klf4, and c-Myc, and induce them in vitro into iPS cells, and lay the foundation of establishing iPS cell lines of Tibet minipig.Method:Get a 35-day embryo of a Tibet minipig. Culture PEFs of the pig isolatedly by enzyme digestion. According to standard protocol from Invitrogen, lentivirus carrying EGFP gene were produced, followed by infecting PEFs by virus supernatant fluid, then confirming the successful production of lentivirus harboring EGFP after 24-48h under the fluorescent stereo microscope.Result:Successfully isolatedly culture the PEFs of the Tibet minipig and the ones of the pig lentivirus high-effectively inflected carrying EGFP, according to the standard protocol from Invitrogen.Methods:template by pLenti-EFla-X-IRES-EGFP(X:Oct4, Sox2, Klf4 and C-myc respectively), PCR amplify target genes(2.35Kbp,2.55Kbp,2.7Kbp,2.8Kbp), clone into vector of pfUGW. Transform products. Select single colonies the next day. Extract plasmid, and identification by enzyme digestion in parallel. Name the vector established pFUXGW(X:Oct4, Sox2, Klf4 and C-myc respectively). Following getting the pFUXGW, lentivirus pack due to protocol from Invitrogen and confirm the lentivirus produced successfully. Establish virus infecting system carrying Oct4, Sox2, Klf4, C-myc and EGFP lentivirus 293FT cell and PEF.Result:Verify established pFUXGW(X:Oct4, Sox2, Klf4 and C-myc respectively) by enzyme digestion. virus supernatant fluid carrying Oct4,Sox2,Klf4,C-myc and EGFP Infect PEFs high-effectively.Method:Infect PEFs with lentiviral supernatant fluid. Abandon the infected fluid after 48h. Change the ES cell medium. Change the medium every day until the colonies become big enough to be picked up to be extensively cultured around day 20 after infection. Assay for the AKP dyeing biological characteristics.Result:Virus shape changed after infected with PEFs. Clone shape appear on the seventh day. Identification of the AKP of all PEFs are all negative. 4.1 The Tibet minipig are new kinds of experimental animals. The Tibetan miniature porcine embryonic fibroblast successfully produced in this experiment provides materials for establishment of ips cell lines of Tibet minipig, and at the same time, it provides nuclear donors for the preparation of transgenic colonies by transgenic somatic nucleus transplantation technique.4.2 Establish successfully the lentivirus-mediated in vitro infecting system aiming at Tibetan miniature porcine embryonic fibroblasts. Lay the foundation for the introduction of different target genes into Tibetan miniature porcine PEFs with the help of the lentivirus. Nucleus from PEFs with foreign genes can be used to produce clone Tibet minipig by transgenic nucleus technology, and then establish the corresponding human disease animal models.4.3 Successfully produce the virus vector with Oct4, Sox2, Klf4 and c-Myc high-effectively infect PEFs. Lay the foundation of the establishment of the Tibetan miniature porcine iPS cell line.