A Study Of IGF-1Transgenic Goat Producrd By Nuclear Transfer Technology

Nowadays, the development of dairy goat industry in Chinese is slow and lacks of international competitiveness, especially in goat milk production. The milk yield of one goat is less than300kg each year, which decreased the economic benefits. To solve this problem, our study tries to improve milk yield with transgenic technology. Transgenic technology is a promising strategy to enhance the performance of mammary glands. An essential component of breeding favorable varieties is selecting a right target gene. Insulin-like growth factors-1(IGF-1) can stimulate cell growth, reproduction and regeneration, and further influence the secreting cells of the goat mammary gland, thus it is an ideal choice as a target and chosen to produce IGF-1transgenic goats. Firstly, we constructed a mammary gland specific expression plasmid pIN with the method of molecular biology. Then plasmid pIN was transfected into the Bcap-37cell line and goat mammary epithelial cells to validate its function in expressing goat IGF-1. Furthermore, the plasmid was injected into goat mammary gland to convince its bioactivity of expressing IGF-1. After analyzing the bioactivity of plasmid pIN, we transfected it into goat fetal fibroblasts and goat ear skin fibroblasts by liposome. Using neomycin antibiotic selection, we picked up two IGF-1-positive clone cells. Then we transplanted the GF-1-positive clone cell into enucleated oocytes through the methods of somatic cell nuclear transfer. When the oocytes were activated and came into the blastocyst stage, we transplanted them into surrogate ewe and obtained five cloned dairy goats. Lastly, we extracted the genomic DNA of clone goats to confirm that these clone goats were IGF-1transgenic goats, using PCR and Southern-blot. At the same time, we detected the copy number and the flank sequence of IGF-1transgenic goats with real-time PCR and TAIL-PCR. Overall, our results cultivated4transgenic goats and laid the foundation for increasing goat milk yeild by transgenic technology.The main experiments were divided into the following five parts. 1. Construction of mammary gland specific expression plasmid pIN and its expression in vitro and in vivoTransgenic technology provides an opportunity to enhance the performance of IGF-1expression, and then modify mammary gland function. This study aims at constructing a mammary gland-specific expression vector, pGN, and validating its function in expressing goat insulin-like growth factor1(IGF-1) both in vitro and in vivo. The backbone plasmid pBCl contained goat β-casein5’ arm and β-casein3’arm, which can express gene specific in mammary gland. Firstly, the igf-1gene was amplified from liver tissue harvested from a Saanen dairy goat and inserted into the downstream of β-casein5’arm. Then the neo gene was cloned from plasmid pCDNA3.1and placed to the downstream of β-casein3’arm as a positive selection marker. In order to analyze the bioactivity of the pIN plasmid, pIN was transfected into the Bcap-37cell line and goat mammary epithelial cells, coupling with goat mammary gland injection. In vitro experiments not only proved that the expression of IGF-1protein and mRNA in transfected Bcap-37cells was higher than that of the control group, but also confirmed that mammary gland specific expression plasmid pIN could be induce to express IGF-1on goat mammary epithelial cells. In vivo studies showed that the expression of IGF-1in pIN injected group was significantly higher than that of the control group. Together, these results strongly demonstrated that the pIN plasmid was constructed correctly and exhibited favorable bioactivity in efficiently expressing IGF-1both in vitro and in vivo, which laid a foundation for increasing milk production.2. Enhancement of gene transfer efficiency in the Bcap-37cell line by amphiphilic molecules (dimethyl sulphoxide and menthol)Simply and efficiently transfer gene into nucleus will facilitate the progress of positive cells screening in producing transgenic animals. One promising method of fast gene delivery is to apply penetration enhancers. Amphiphilic molecules, such as dimethyl sulfoxide (DMSO) and menthol, could serve as non-toxic vehicles in improving gene transfer efficiency. In this study, the cytotoxic effects of DMSO and menthol were evaluated using MTT assays. Gene delivery efficiency in a human breast cancer cell line (Bcap-37) was investigated by quantitative PCR, fluorescence microscopy and flow cytometry. Results proved that non-toxic concentrations of DMSO (2%, V/V) and menthol (12.5μM) enhanced the efficiency of liposome-mediated gene delivery in Bcap-37cells. Quantitative PCR results showed that the expression of growth hormone (GH) in post-menthol and pre-DMSO treatment groups were10times as that of the liposome group, while in the pre-menthol and post-DMSO treatment groups, a30times increase in GH mRNA expression was observed. Both DMSO and menthol treatments increased the numbers of cells expressing green fluorescent protein, which was shown by fluorescence microscopy experiments. Compared to the liposome group, the number of positive cells in the pre-menthol and post-DMSO treatment groups was significantly increased by15%. Furthermore, cell cycle analysis demonstrated that there were significant differences among the DMSO-treated group, the menthol-treated group and the normal group, which implied different effects of DMSO and menthol treatments. In conclusion, both non-toxic and harmless DMSO (2%) and menthol (12.5μM) treatments improved gene transfer efficiency, and post-DMSO treatment may be the most effective protocol in increasing gene transferring efficiency.3. Improvement gene transfection efficiency in the Bcap-37cell line with NLS and SPB-NLSLow transfection efficiency severely blocks the development of transgenic animals. The process of exogenous DNA fragments entering nucleus is a major rate-limiting step, especially for the large DNA fragments. Nuclear localization sequence (NLS) peptide mediates the trafficking of nuclear protein, from cytoplasm into nucleus. Succinimidy-4-(psoralen-8-yloxy)-butyrate (SPB) can non-covalent couple with DNA molecules to prevent DNA from degradation when delivered into cell. Peptide "CGGPKKKRKVP (classic NLS)" and peptide derivative "SPB-PKKKRKV" were synthesized to mediate transfection in vitro, aiming at improving large DNA fragments transfection efficiency, especially for producing transgenic animals. To explore their biology function, we compared GH mRNA and GFP protein expression by qRT-PCR and flow cytometry. Results identified NLS group (increased by69%) and SPB-NLS group (330%) significantly improved the expression of GH mRNA. Likewise, SPB-NLS group increased the number of GFP positive cells (32.4%), but NLS group decreased the number of GFP positive cells (75%). Further analysis (western blot) demonstrated the function of SPB-NLS in hard-to-transfect Bcap-37cell (increase the expression of GFP) and target GMEC cells (improve the expression of IGF-1). In conclusion, SPB-NLS served as a transfection enhancing agent, can be widely used in both nuclear delivery and producing genetically modified animals. 4. The screening of IGF-1-positive clone cells and the production of IGF-1transgenic goatsThe production of transgenic animals has always been the focus and difficulty, especially for large-scale livestock (such as goats and castles). In this study, we firstly isolated and cultured dairy goat fetal fibroblasts and ear skin fibroblasts. Then we explored the optimal concentration of G418for positive-cells screening. Results displayed that when the G418concentration came to800ng/mL, fetal fibroblasts would be killed within two weeks. In terms of the ear skin fibroblasts, the optimal G418concentration was600ng/mL. After confirmed the optimal concentration, we transfected mammary gland specific expression vector pIN into goat fetal fibroblasts and ear skin fibroblasts. In the following of10-14days screening, we picked up monoclonal cell mass, then cultured them in48-well cell culture plates and dropped the concentration of G418to300ng/mL. The picked monoclonal cells were cultured until full of a6-well cell culture plates. Then the monoclonal cells were divided into two parts. One part was used to extract genomic DNA for identify, the other part was frozen for nuclear transplantation. Our study finally screened46monoclonal cell lines. From these monoclonal cell lines, we selected12monoclonal cell lines with good growth state by PCR identification. The PCR results showed that there were2positive clones in the cell’s genome which IGF-1gene has integrated into. After identification, we transplanted the IGF-1-positive clone cell into enucleated oocytes through the methods of somatic cell nuclear transfer. At last, we obtain4IGF-1transgenic goats.5. The identification of IGF-1transgenic goatsTransgenic technology has recently been employed to create animal lines with new genes inserted into their chromosomes. The genome of transgenic animals comprised of exogenous gene, specific promoter, regulatory elements and marker genes, which were the basis for the identification of transgenic animals. The identification of transgenic animals contained two parts. The first part was detecting the presence and the expression of the exogenous gene, the second part was analyzing the integrity site and the copy number of transferred gene. In this study, we firstly tried to prove the clone goats to be IGF-1transgenic goats by PCR and Southern-blot. Then we used the methods of real-time PCR and TAIL-PCR to analyze the copy number and the integrity site of transferred IGF-1gene. PCR results demonstrated that the igf-1gene and neo gene were integrated into the genome of IGF-1transgenic goats. Further experiments (Southern blot) proved that these clone goats were IGF-1transgenic goats. Absolute quantitative PCR could be used to precisely analysis the copy number of exogenous gene. To begin with, we established the standard curve (log2N (copy number)=-1.0244△Ct+5.3576(R2=0.9963)), then detected the copy number of four IGF-1transgenic goats. Results showed that the four IGF-1transgenic goats contained8copy IGF-1gene in their genome. At last, we attempted to identify the integrated site of exogenous IGF-1gene. TAIL-PCR results identified4specific integrating sites which were listed in bovine chromosome2,11,16and18. Overall, our study initially established a system about detecting the copy number and the integrating site’s flanking sequences of exogenous gene, which laid a solid foundation for the genetic research of transgenic dairy goats.