Production Of GH Transgenic Goat By Somatic Cell Nuclear Transfer

There are more than 190 countries raising dairy goats around the world, and China has the largest dairy goat herds. However, the level of large-scale farming for dairy goats is still low in China, as well as the annual milk production of individuals generally. Hence, they have become urgent need for improving breed of dairy goat and raising dairy milk production of individuals. Growth hormone (GH) plays an important role in both mammogenesis and lactating process. Additionally, GH can also maintain the number of mammary epithelial cells during lactation, resulting in increasing of milk production. Therefore, GH as the target gene was selected and produced the GH transgenic dairy goats with high milk production by somatic cell nuclear transfer technique.Firstly, β-LG gene promoter element was used to construct the vector pcGH. The pcGH could specifically expressed GH in the mammary gland by molecular cloning technique, and was transfected into mammary epithelial cells which had been isolated and purified, aiming at verifying the expression of GH in vitro. Secondly, the linearized vector pcGH was transfected into the isolated and cultured goat fetal fibroblast cells by electrotransfection method, and then transfected GH gene-positive cells that picked out by G418 drug screening. These positive cells were further transplanted into the enucleated oocytes. Cell fusion was induced by electrical pulse which also activated the oocytes. After developing to blastosphere or morula blastocyst, the transplanted oocytes were transplanted into the uterus of surrogate ewe to produce FO generation of cloned goats. Thirdly, the integration of exogenous gene in genome was examined by general PCR, Southern blot, Real-time quantitative PCR and thermal asymmetric interactive PCR (TAIL-PCR). Finally, the growth, physiological conditions, breast development and milk production of FO generation GH-transgenic dairy goats were detected to assess the effects of GH gene for dairy goats; while the growth and physiological conditions of lamb which fed with milk from FO generation GH-transgenic dairy goats were also detected to assess the security of GH-transgenic dairy goats.This study hopes to obtain transgenic dairy goats with high milk production and is divided into the following four parts in detail.1 Construction of goat mammary gland-specific vector pcGHThe purpose is to construct a mammary gland-specific expression vector, pcGH, and demonstrate effective GH expression in isolated goat mammary epithelial cells in vitro by ELISA. Firstly, to construct the mammary tissue-specific expression vector pcGH, the 5’ and 3’flanking regulatory sequences of β-LG were cloned from the genomic DNA using a DNA extraction kit. Based on the genomic DNA sequence of goat β-LG, the 5’flanking sequence, including 5’regulatory regions to whole exon 1 of β-LG, was amplified using the primers ZP1 (5’end Xho I) and ZP2 (5’end EcoR I); the 3’flanking sequence, including partial exon6, whole exon7, the polyadenylation sequences and 3’genomic fragment of β-LG, was cloned using the primer pair ZP5 (5’end Sal I) and ZP6 (5’end Sal I). And total mRNA was harvested from the pituitary gland of a Saanen goat. The cDNA encoding goat GH was cloned using an RT-PCR method. The primers contained an EcoR I recognition site in the 5-terminal end of ZP3 and a Sal I recognition site in the 5-terminal end of ZP4. Thirdly, the 5’flanking sequence of β-LG (p-LG5) was digested with. Xho I and EcoR I and inserted into Xho I site of vector pV, named pV-BLG5. Then, gh gene fragement and pV-BLG5 digested with EcoR I and Sal I were linked, named pV-BLG5-GH. At last, the 3’ flanking sequence of of β-LG (β-LG3) was digested by Sal I and subsequently inserted into linearized pV-BLG5-GH, generating the final recombination vector pcGH. The pcGH vector was identified by PCR amplification and subsequent restriction enzyme digestion, which indicated that the recombinant vector pcGH was constructed successfully. To investigate whether GH could be expressed in the mammary gland, we isolated and cultured goat mammary epithelial cells through tissue culture, which were identified by immunohistochemical methods. The vector pcGH was transfected into goat mammary gland epithelial cells with liposomes (Lipofectamine 2000, Invitrogen) according to the manufacturer’s protocol. The supernatant of each well was collected at 48 h,60 h and 72 h after transfection. ELISA results revealed that GH expression in transfected with pcGH goat mammary epithelial cells and reached the highest level at 48 h and decreased at 60 h and 72 h. GH expression in transfected goat mammary epithelial cells was significantly higher than in non-transfected goat mammary epithelial cells at 48 h (P< 0.01),60 h and 72 h (P< 0.05). These results laid a solid foundation for foster GH trsansgenic goat improving mammogenesis and lactation performance in future.2 Screening of transfected GH gene-positive cells and Preparation of GH-transgenic dairy goatsThis section views to isolate and culture goat fetal fibroblast cells, transfect and screen transfected GH gene-positive cells, obtain oocytes by superovulation and produce GH-transgenic dairy goats by somatic cell nuclear transfer technology. Firstly, we isolated and cultured goat fetal fibroblasts cells through tissue culture, and identified them as females by PCR. Then the linearized vector pcGH was transfected into the goat fetal fibroblast cells by electrotransfection method. After transfection 48 h, the medium was changed and added G418 (500 μg/mL) to carry out resistance screening, cloning mass appearing on the 12th day approximately. Monoclonal cell masses were picked out and passaged in 48-well plates, and collected cells until growing to 80-90% under the condition of halving G418 concentration. A total of 65 transfected GH gene-positive cells were identified by PCR. After starvation, these positive cells were cryopreserved as the nuclear transfer donor cells. Secondly, we obtained 388 oocytes which were from 37 ewes by superovulation, serving as the nuclear transfer recipient. Specifically, after nuclear transfer, the numbers of available oocytes was 337; the number of fusion eggs was 264; the fusion rate was 78.34%. while after activation, there were 254 reconstructed embryos,115 of which were directly transplanted into the oviduct of 17 recipient goats and the other 138 reconstructed embryos were embedded in agar and then transplanted into the oviduct of recipient goats, cultured in vivo for 5 days. Then we recoveried 126 embryos, and 64 of which developed to the blastocyst stage. These embryos were transplanted into the uterus of 39 transplant recipient ewes,1-2 per transplant. Totally, there were 56 transplant recipient goats and 27 of them were pregnant on the 35th day. Finally, we got six surviving cloned goats, which laid the foundation of identification for GH-transgenic dairy goats.3 Identification of GH-transgenic dairy goatsThis section focused on identifying and analyzing the integration of exogenous gene in the F0 generation GH-transgenic dairy goats. Firstly, three pairs of specific primers were used to detect the integrated exogenous gene by general PCR. Secondly, southern-blot method was used to futher detect the integration situation in order to exclude PCR false-positive interference. Finally, we confirmed the copy number and integration site of GH gene by real-time quantitative PCR and TAIL-PCR. Additionally, junction PCR verified the integration site. The results of PCR and Southern-blot indicated that GH gene was successfully transferred into all the cloning goats; the results of real-time quantitative PCR showed that the copy numbers of GHcd-2 and GHcd-7 GH-transgenic dairy goats were 12.95 ± 0.18 and 12.24 ± 1.12 respectively; and the results of TAIL-PCR test revealed that the exogenous genes of GHcd-2 and GHcd-7 GH-transgenic dairy goats were integrated into chromosome 3 and chromosome 11 genome respectively. The above results provide genetic foundation for the safety evaluation of GH-transgenic dairy goats.4 Preliminary safety assessment of GH transgenic dairy goatThis experiment studied on the self-security of GH-transgenic dairy goats and safety of GH-transgenic dairy goat milk. Firstly, we researched the effects of exogenous gene on the growth and development of dairy goats through examining the growth and development performance of F0 generation GH-transgenic dairy goats. The results showed that no significant difference existed in the growth and development between the F0 generation GH-transgenic and non-transgenic dairy goats (P> 0.05). Then the blood routine index, blood physiological and biochemical indicators of F0 generation GH-transgenic dairy goats were detected to research effects of exogenous gene on goat physiology. The results indicated that the various physiological and biochemical indexes of F0 generation GH-transgenic dairy goats were within the normal range, showing no difference with normal goat milk (P> 0.05); while from breeding to latter half of gestation, the breast of F0 generation GH-transgenic dairy goats developed significantly faster than that of normal dairy goats, and the udder depth (UD), udder width (UW), udder circumference (UC) and distance between teats (DT) of GH-transgenic dairy goats were significantly larger than that of normal dairy goats (P<0.01). In the early lactation period, the milk production of GH-transgenic dairy goats was higher than that of normal dairy goats (P<0.05). The milk composition analysis revealed that no significant difference existed in the milk composition between GH-transgenic and GH-transgenic dairy goats dairy goats. Namely, GH-transgenic dairy goats had an increase in the milk production without change in milk composition. Additionally, Western-blot analysis showed high expression of GH in milk, demonstrating that constructed vector was capable of expressing GH protein in the mammary gland. Finally, we fed lambs with the milk of F0 generation GH-transgenic dairy goats, and tested the growth and physiological and biochemical indicators of lambs, finding no influence in the growth, development and physiology of lambs (P> 0.05). This study initially obtained GH-transgenic dairy goats which had significant increase in milk production in the early lactation period, and conducted a preliminary assessment for the safety of F0 generation GH-transgenic dairy goats and their milk, our studies provided certain references for breeding high milk production transgenic dairy goats in future.