Production Of Transgenic Goats Expressing Alpha-lactoalbumin By Somatic Cell Nuclear Transfer

The technology in health care and animal husbandry have broad application prospects, which is by somatic cell cloning to produce transgenic goats, and then create the goat mammary gland bioreactor, cultivate the new materials of transgenic goats. This paper focuses on the establishment of an effective, economical technology platform for transgenic cloned goat, which carried out the following test for goat oocytes in vitro maturation and developing the key techniques of alpha-lactalbumin transgenic cloned goats.Experiment1: To summary the statistics of ovaries obtained from the slaughterhouse for the different months in the region a valid number of oocytes obtained from each ovary and the affection of oocytes in vitro maturation for different months in the region. The showed that: each ovary which collected the average number of oocytes during October, November, December and January can be higher they are1.957,1.938,1.804and2.525oocytes per ovary. In these months, there is no significant difference between March, April, May, June, October, November, December there is significant difference between January and March, April, May, June, but no significantly difference between January and October, November, December. And the rate of oocyte maturation is higher during October, November, December and January, which respectively is:55.37%,53.74%,56.61%,55.71%. In these months, there is no significant difference between March, April, May, June, October, November, December there is significant difference between January and March, April, May, June, but no significantly difference between January and October, November, December. Overall, the ovarian which collected around autumn and winter had better quantity and quality, compared to the other months. That showed oocyte collection and oocyte maturation were better during January, October, November and December in the region, which more suited to test.Experiment2: To explore the effect of oocyte maturation medium supplemented with vitamin C on oocyte in vitro maturation. The results showed that: Adding500μM,1000μM Vc groups were significantly higher than The control group (P <0.05). Among them, the maturation rate of the group of adding1000μM Vc was the highest (68.18%). Therefore, it could significantly promote oocytes maturation if adding the concentration of1000μM Vc in the maturation medium.Experiment3: To investigate the effect of oocyte maturation medium supplemented with human basic fibroblast growth factor on oocyte in vitro maturation. The results showed that: the maturation rate of oocytes and parthenogenetic embryo cleavage rate of the experimental groups were higher than the control group, but there was no significant difference (P>0.05). The best result of them was the50ng/mL hbFGF group, and the maturation rate was70.43%, the cleavage rate was68.49%. Therefore, the suitable concentration of hbFGF can promote in vitro oocyte in vitro maturation and post-development.Experiment4: Compared of the different fusion parameters on the fusion rate of reconstructed embryos. The results showed that: parameter with1.40kv/cm,30μs,2DC, more effective integrating reconstructed embryos, the fusion rate was68.99%, which was not significantly different from1.40kv/cm,30μs,1DC,120kv/cm,30μs,2DC groups, but was significantly different from the group of120kv/cm,30μs,1DC.Experiment5: The pBC1-N-Lact mammary specific expression vector was constructed by the target gene of cloned mouse α-lactalbumin cDNA and the skeleton which is transformed by PBC1-Neo vector. Digestion and sequencing results showed that: we have successfully cloned α-lactalbumin gene, and got the correct expression vector backbone. And that the mammary gland specific expression vector for a positive goat fetal fibroblasts. We have successfully got the goat fetal fibroblast cells with mammary gland expression vector positive by liposome transfection.Experiment6: We have established a relatively stable platform for the production of transgenic goats by the preparation and exploration of above five tests. We have producted about220embryos by somatic cell nuclear transferring which use the Huang-Huai white goat fetal fibroblasts as donor cells with α-lactalbumin gene. The embryos were transplanted to10receptor goats’ wombs. And then two lambs were born after full-term pregnancy. After identification, one is goat with α-lactalbumin gene, the other is the cloned goats. The results showed that: we have successfully produced transgenic goats by somatic cell nuclear transfer.In summary, we have got the first successful case of transgenic cloned goat with milk quality regulation genes in our laboratory, which marks we have made important breakthroughs in the field of transgenic animal research in Anhui province. And we also have established a basis for exploring the establishment of transfer cloning technology with independent intellectual property rights, and developing transgenic cloned goat with LA gene.