The Studies Of Somatic Cells Purification, Human COL1α1 CDNA Transfection And Nuclear Transfer Embryos Development Of Dairy Cow

Animal mammary gland bioreactor, also called individual animal mammary gland expression system, is the use of regulatory sequences of milk protein gene to construct expression vectors, produce transgenic animals and guide specific foreign genes expression.It has the advantages of high output, low cost, easy purification and high biological activity et al. Currently transgenic mammary gland bioreactor have been successfully used in clinical treatment and many other related fields. Transgenic cloning technology can screen genetic modified gene at cellular level, ensure that individuals are genetically modified cloned animals.In higher animals, collagen is widely distributed in connective tissue, such as skin, bones, organs mesenchymal cells, muscle cavity, ligaments and sclera et al. Cornea is entirely made of collagen. Collagen is an important structural proteins, play the function of supportive organ and protect the organism. Because of its high biological activity and low immunogenicity, in the areas of pharmaceutical, medical, beauty product, food industry, collagen have been widely used. Human collagen, in particular procollagen is more important for human to use, but because of ethical and moral constraints, it can not directly access. So, produce human collagen using mammary gland bioreactor approach is necessary, feasible and safe.Using mammary gland bioreactor to produce human collagen, first is to obtain collagen gene, then transfer this gene into the mammary gland expression vector. Second is to transfer the vector to target cells and obtain the positive target cells that is genetic stability and can be unlimited amplification. At last, using somatic nuclear transfer technology to obtain positive embryos.For the purpose of obtaining the mammary gland gene expression vector, experiments were firstly extracted mRNA from human foreskin, reverse transcription to obtain one of the collagen protein cDNA fragment. In the 3'end of the cDNA connect a 6×His protein separation tag. Then using pBCl plasmid as based vector, using (3-casein gene promoter as a regulatory element, reconstructed the expression vector that connect with human type I collagen al cDNA gene. Finally, with enhanced green fluorescent protein (EGFP) and neomycin resistance gene (Neo') as a double marker genes into expression vector, completing the transformation of mammary specific expression vector. Synthesized cDNA fragments were sequenced. Blast results showed that the Chinese cloned type I al collagen cDNA gene are very similarity comparision with Gene Bank. The fragments of the vector were amplified by PCR analysis,the result showed that the constructed vector structure was complete and correct.In order to obtain efficient transfectant of monoclonal cell lines, experiments established fibroblasts, cumulus granulosa cells and oviduct epithelial cell lines. These target cells were carried out liposome transfection and electroporation transfection. The results showed that fibroblasts and cumulus granulosa cells have been successful transfected by the method of liposome transfection, while oviduct epithelial cells is failed. But for the oviduct epithelial cells, after carrid out electroporation transfection, obtained 20.8% of transfection efficiency on the condition of 90 mOsmo / kg hypotonic buffer and 800V voltage electric tension (2mm electroporation tube). The three types of cells use G418 that concentration at 800μg/mL can effectively screen out the positive cells. The primary screened cells use EGFP as marker can further screen out transgenic positive clones that comes from one single cell and the monoclonal cell lines have the same genetic background.In order to understand the apoptosis of fibroblasts, cumulus granulosa cells, oviductal epithelial cells and mammary epithelial cells that cultured in vitro, the methods of flow cytometry are performed. The results showed that at the same growth cycle, once the cells at the condition of contact inhibition or plateau phase, the process of apoptosis is significantly faster. Also with the increase of cell generations, the apoptosis rate increased. Overall, the rate of apoptosis in mammary cells is the fastest, followed by the oviduct epithelial cells. Cumulus granulosa cells' apoptosis rate was lower than fibroblast before 15 generations, but after 15 generations the other way around.Finally, using the genetically modified cells as donors conducted nuclear transfer (NT) and the reconstructed embryos were compared. The comparison found that:1) The reconstructed embryonic development was no significant difference (P>0.05) between the different transgenic cell lines of fibroblasts; 2):Transgenic and non-transgenic cells as NT donors, their fusion rate has shown a significant (P<0.05) decrease, but their cleavage rate and morula/blastocyst rate was not significant; 3) Three types of genetically modified cells as NT donors, the results showed that the fusion rate of fibroblasts and cumulus granulosa cells was significantly (P<0.05) higher than the oviduct epithelial cells. However, the morulae/blastocyst formation rate of fibroblasts was significantly lower than cumulus granulosa cells and oviduct epithelial cells after fused (P<0.05). These results indicate that:1) Different transgenic lines of the same type of cells probably have different insertion site and copy number, but the early embryonic development had no significant difference; 2) Transgenic process and the selection process can give rise the membrane structure change to a certain extent and resulting the fusion rate was poorer; 3) In the process of fusion, oviduct epithelial cells was relative difficulty compared with fibroblasts and cumulus cells, but in the process of reconstructed embryonic early development, cumulus cells and oviduct epithelial cells have higher viability than fibroblasts.This thesis had a series of systemic study that include vector construct, cells transfection, monoclonal cell lines amplification and the genetically modified embryonic development laws. The finally purpose is to provide useful reference for the development of mammary gland bioreactor.