Isolate And Culture Porcine Embryonic Stem Cells And Screen OCT4-eGFP Tracer Cells

Embryonic stem cells are pluripotent cells that have the self-renew ability to proliferateindefinitely and to differentiate into three germ layers in vitro. Chimeric experiments show thatembryonic stem cells have the ability to chimeric into embryoid bodies, which is the uniquecharacter of embryonic stem cells. This application of the characteristic can be fitted to generategenetically modified animals. Genetically modified animals had a wide range of applications inbasic medical research, drug experiments, disease research. Currently, mouse, rat, and humanembryonic stem cell technology has been very mature, but which of this in dometic animals is notmature yet. The pig is a good animal model for human disease and an ideal material forxenotransplantation. Therefore, it is necessary to develop the suitability of the pig ESC cell cultureconditions.We studyed the early porcine vitro embryo development and increased the adherent rate ofblastocyst by droping medium in per hole and compared the effect of two kinds of culture medium:two factor medium and four factor medium. We discuss the possibility of establishing the genuineporcine embryonic stem cells. We used TALEN techonology to improve the probability ofhomologous recombination and inserted2A peptide sequence with EGFP and neo with loxpsequence in the stop codon of Oct-4gene exon5under the endogenous Oct4promoter-driven Oct4and eGFP expression. Throught using somatic cell nuclear transfer technology could acquireblastocysts which can accurately simulate the endogenous Oct4expression. We could able toobserve whether the pES derived from early vitro embryo expressing the Oct4gene thought theGFP fluorescence. It is benefical for pES pluripotency identification and cultivation systemoptimization, and then determined the pluripotency of cell. The main experimental methods andresults are as follows:The first part, we collected the porcine vitro embryo to observe morphology and measurediameter. The rezult show a positive correlation between the age and the diameter for the earlystage porcine vitro embryos. We also found the embryos of the same age had differentdevelopmental stage between somatic cell nuclear transfer embryo and parthenogenetic embryo.So it was not scientific to judge porcine early embryos development by age only. We attempted toisolate porcin embryonic stem cell from early vitro embryo. We compared two sets of culturemedium: we derived epithelioid colony from culture medium of double factor and pass threepassages by mechanical methods, but by RT-PCR results for the expression of Cdx2gene, but didnot express Oct4gene. But the culture medium of multi-factor get2small colonys, colony had no proliferation after passage.The second part, we established the porcine ear fibroblast cell line. The drug pressure curveshow that300μg/mL was the appropriate screen concentration. Though co-tansfection of Donorplasmid and TALEN mRNA into ear fibroblast cell WPF17-1and WPF17-2, we acquired4positive cell clonys by PNS. The experiment has acquired OCT4-eGFP knock-in cells.