Studying On Transgenic Dairy Beta-glycosidase Specific Expressed In Mammary Gland

1.Transgenic animal research has become one of the most effective ways for the development of life science and new animal husbandry. This study integrated the Beta-glycosidase (LacS) to the cow genome through the construction of mammary gland specific expression vector and expressed it in the transgenic individuals so that to reduce the proportion of lactose in milk.The promoter sequence of cytomegalovirus (CMV) gene was derived by Polymease C hain Reaction(PCR) amplification from the plasmid pEGFP-Cl and sites Sad and KpnI were introduced at each of the two ends of the sequence, respectively. Then the promoter CMV fragment was assembled into the linearized plasmid pDsRed2-l that had been digested with SacI and KpnI. The resulted vector which contains an ERFP coding sequence in downstream of a CMV promoter was named pDsRed-CMV.A LacS gene fragment was synthesized according to the sequence of LacS gene from Sulfolobus solfataricus using the preferred codons of cow. The codon-optimized LacS gene together with the poly (A) sequence fragment amplified from the cow blood genome were assembled into the MCS of the plasmid pDsRed-CMV. The resulted vector was named pDsRed-CPL. The promoter sequence of casein (BC) gene was derived by PCR amplification from the cow blood genome and sites Xho Ⅰ and Sal Ⅰ were introduced at each of the two ends of the sequence, respectively. Then the promoter BC fragment was assembled into the linearized plasmid pDsRed-CPL that had been digested with Xho Ⅰ and Sal Ⅰ. The resulted vector which contains ERFP gene and a LacS gene coding sequence in downstream of a BC promoter was named pBL. Endonuclear enzyme digestion and PCR amplification proved that the construction of vector pBL succeeded.2.The bovine fetal skin fibroblast (BFFB) cells were isolated by attachment of tissue pieces from skin gestation bovine fetal. The cells, isolated as primary fibroblasts from gestation fetal bovine skin, were frozen and stored. Ten bovine fetal skin fibroblast cell systems were established, three of which were cultured for drawing the growth curves and nuclear analysis. The results suggested that F5, F10and F15generation cell growth of the three cells were experienced the latency in the first0-2days, the logarithmic growth phase in the next3-6days and plateau phase7days. All of them maintained diploid karyotype with a ratio of more than80%. So the bovine fetal fibroblast cells before the15th generation gave a good fit to transgenic cloning manipulation.3.The correct plasmid (DS-Red-LacS2) was transferred into the selected three bovine fetal fibroblasts using the liposome mediated transfection method. The cells expressed red fluorescent after transfection forl8-24h were diluted (1:30) and cultured until50%-70%of the cells grown into confluence, after which G418selection (600μg/ml) medium was applied until the formation of monoclonal. Mixed the clones expressed red fluorescent for expanding culture and detected the target gene. The results indicated that target genes can be detected in the hybrid clones expressed red fluorescent. Thus screened hybrid clones can be used as a nuclear donor cells.4.LacS gene transgenic embryos was constructed using somatic cell nuclear transfer method. The culture and developmental parameters of the transgenic embryos were determined based on the study of oocyte parthenogenetic development system. With the best fusing conditions of voltage100v, pulse time30μs and twice pulses, the fusing rate reached78%-80%. Inomycin plus6-DMAP was used for activation with the parthenogenetic activation rate of90%and blastocyst rate of30%.5.No-surgical technique was used for LacS gene transgenic embryo transfer. Compact morula, blastocyst and early blastocyst were transferred simultaneously into the uterus of Estrus synchronization receptor cow. The test results after60days of pregnancy showed that reconstructed embryos derived from2different cell systems as a nuclear donor had receptors in bovine pregnancy after implantation. The embryo originated from the P06.17cell as nuclear donor had higher pregnancy rate and can successfully birth newborn calf. PCR was carried out to detect the target gene with the genome from newborn bovine jugular venous blood as template. Genetic tests were positive, which proved that the newborn calves were LacS transgenic cloned cattle.