The Research Of The SCNT Technology To Produce The Human B-Defensin-3 Gene Of Bovine

Defensins is a kind of endogenous antibiotic which existed extensively inside of plant corpus and animal body color as well as insect, its function that is sterilization broad spectrum, it suppose to be an important part of nature immunity in Organismal Biology specialization. It has a stable antibacterial activity and low toxicity, which can eliminate and reduce bacterial resistance and drug resistance. Therefore, we pay more attention to its medicinal value. Today, because the abuse of antibiotics, many experts had made use of defense, which has always been to replace the role of antibiotics. And, through genetic engineering methods to produce large quantities of defensins will be better for the benefit of mankind.The purpose of this study is to transfer production of transgenic cattle defensins, based on previous experiments, the research is that:By isolating adherent tissue culture of bovine fetal fibroblast cells and epithelial newborn bovine fetal fibroblast, and compare to granule cells, then use the method of power transfer put the defensin gene into genome of the three cells. Then compare the number of monoclonal respectively, the best choice for body is that using data on bovine fetal fibroblast cells.Using Lipofectamine transfection method and the electric transfection bovine fetal fibroblast cells respectively, and compare the effects of different transfection efficiency of transgenic, the result of it that the method of electronic transfer of data is more efficient.Respectively, after cell transfection and G418 selection for 4 weeks of bovine fetal fibroblast cells and non-transfected in vitro for 4 weeks in the same fibroblasts as nuclear transfer donor cells, then compare the fusion rate, cleavage rate and blastocyst rate. Through the data we found that after G418 selection of transfected cells 4 weeks of reconstructed embryos with non-transfected cells were compared with the control group, fusion rate, cleavage rate were not significantly different, However, transgenic blastocyst rate was significantly lower than control group. This shows that gene transfer and selection process get adverse effects on the development of transgenic embryos.Transfer somatic cell nuclear into the different diameters of the donor cell respectively, through the data we found that donor cells and the diameter of the reconstructed embryo fusion, cleavage and blastocyst development consequences. With a diameter of 15-20μm of the cells of cloned cells for embryo cleavage rate and blastocyst rate was significantly higher than the diameter of less than 10 or greater than 25μm of the cells for the cells of cloned embryos.Respectively, by using the method of serum starvation with the contact inhibition and growth of cells treated with strong cell donor cells, In the experiment, we found that the treatment of donor cells on the morphology of hair follicle cells have great impact. Through the data we found that growing cells do donor cell fusion rate and blastocyst rate were significantly higher than after treatment and contact inhibition in cells treated with serum starvation.By using the method of Pcr and green fluorescent protein positively identify the transgenic embryos, and was used for the embryo transfer.After embryo transfer, we obtained the transgenic cloning cattle which were transfering defensin.