Preparation Of Bovine Transgenic Embryo For Resistance To Mastitis

Mastitis is a common disease in dairy cows which brings out great harm to dairy industry and food.As the drug-resistant bacteria appearing, it is less effective for antibiotic to treat mastitis. It may be analternative new approach to prevent and cure mastitis through producing anti-mastitis transgenic cattle.This study which might lay the foundation to produce the anti-mastitis bovine in the further was carriedto construct transgenic bovine somatic cell nuclear transfer(SCNT) embryos containing Lysostaphin andEndolysin genes through transgenic cells by SCNT,which the oocytes were denucleate by clicking andfused cytoplasm without zona pellucida.Meanwhile,the effects of MG123(Protease inhibitor,Z-Leu-Leu-Leu-al) on SCNT,and on the condition of transfecting the Lysostaphin and Endolysin genesto bovine fetal fibroblast cells were researched.The results were as follows:Experiment1: The study on MG132affected the efficiency of SCNT①The bull fetal fibroblast cell lines through tissuecultivation were established in thisexperiment. The growth curve of the fetal fibroblast cell lines was corresponding with the rhythm ofcell growth in vitro (the curve of S model), the cell activity was high, and the chromosome numberwas normal (2n=60), The cell lines could satisfy the demand of being the donor cell.②The toxic effect of proteasomeinhibitor MG132on parthenogenetic activated bovine embryodevelopment after oocytes treated with MG132were checked.It did not affect the embryo developmentsignificantly（parthenogenetic embryo cleavage rate was73.33%, blastocyst rate was31.82%）thatoocytes were treated1h with the maxi concentration of7μmol/L of MG132,and it did not affect theembryo development significantly（parthenogenetic embryo cleavage rate was69.88%, blastocyst ratewas36.21%）that oocytes were treated2h with the maxi concentration of3μmol/L of MG132.③Thecleavage rates of the reconstructed bovine embryos（SCNT）without zona pellucida were85.54%、73.42%, and the blastocyst rates23.94%、17.24%after treated1h with7μmol/L MG132and2h with3μ mol/L MG132,respectively. There was no significant difference between experiment groupsand the control group(cleavage rate was76.32%, blastocyst rate18.96%,respectively).There was nosignificant difference of the relative active of p34cdc2kinase at0h and2h after fusion between theexperiment groups and the control group.The results showed treatment of reconstructed embryos with MG132did not promote thedevelopment of the SCNT embryosExperiment2: The study on efficiency of exogenous genes transfected to cells and the preparationof the transgenic embryos for resistance to mastitis①Thevectors containing Endolysin and Lysostaphin genes and two other marker genes-EGFPand neo were transfected into bovine fetal fibroblast cells by electrotransfection. The bovine transgeniccell lines of stable transfection which the Lysostaphin and Endolysin genes were integrated to thechromosome of cells detected by PCR were obtained through G418selection. ②Theparameters of AMAXA nucleofaction about bovine fetal fibroblast cell transfection wereoptimized which the parameter of T-016was the most appropriate among the four parameters. AMAXAnucleofaction was appropriate for acquiring the bovine fetal fibroblast cells of stable transfection whichthe transfection efficiency（20.11%）was better than it（3.88%）by BTX electrotransfection.③Thetransgenic SCNT embryos which the Lysostaphin and Endolysin genes wereintegrated tothe chromosome of embryos detected by PCR were acquired and the blastocyst rate was20.08%.