Study On The Establishment Of A Ear Marginal Fibroblast Cell Line And IVF Of The Bactrian Camel Fetal

Embryonic stem (ES) cells are totipotent cells which derived from animal inner cell mass (ICM) or primordial germ cells (PGCs). As a new experimental materials, ES cells are widely used in animal cloning, genetically modified animal production, eukaryotic cells of gene expression and regulation, and the creation of animal models of human genetics, human organ transplant material production and cell differentiation mechanisms, etc. Combinations of ES cell cloning technology and embryo engineering has great theoretical significance and value of the reality to clarify the basic rules of the animal growth, rescue and protect endangered animal genetic resources and establish advanced animal breeding technology system. In this study, we chose bactrian camel for experimental subject, established a Bactrian camel fetal ear marginal fibroblast cell line; manufactured Bactrian camel frozen sperm; researched Bactrian camel ivm and ivf of oocyte; separated and identified preliminarily Bactrian camel ES cells. The following main conclusions were reached:1. The Bactrian camel fetal ear marginal tissue was cultured with explants and trypsinization and then subcultured. Observations and analysis on the morphology, cell activity, dynamic growth, immunohistochemistry, karyotype, fluorescin plasmid transfection and expression, microbial contamination were carried out. The results showed that population doubling time (PDT) of the cells was47.2h; the mode of chromosome number was74; the appraisal result of immunohistochemical was positive for vimentin and negative for cytokeratin; the exogenous plasmid could integrated and express in the cell; detection for microbial contamination was negative.2. Used artificial vagina collecting Bactrian camel semen, and producted frozen semen. The results showed that the effect of formula2was best for the extenders of frozen semen, the effect of Bactrian camel frozen semen was advisable when the balance time is2-4h.3. Acquired Bactrian camel ovariums and colleced oocytes, tested IVM and IVF. The results showed that surface cutting was higher than aspiration and puncture in recovery of Bactrian camel oocytes; the effect of culture medium was best when the amount of the hormone was20μg/mL LH+10μg/mL FSH+1μg/mL E2; TALP fertilization system was better than BO fertilization system in Bactrian camel cleavage rate.4. Used whole embryo culture and enzyme digestion separating the Bactrian camel blastula, and cultured ICM in C57line MEFs and Bactrian camel fetal fibroblast cells. The results showed that embryos adhered and formed primary cell colony easily by whole embryo culture; the effect of MEFs was better than Bactrian camel fetal fibroblast cells; the form of cells was similar to ESc observed with microscope, the result of alkaline phosphatase staining was positive.