Preparation Of TALEN Mediated Trans-HSA Gene Cloned Embryos

Human serum albumin(HSA), one of the most abundant protein in the plasma, has important biomedical value. Traditional production of HSA mainly comes from plasma separation, but it has the risk of transmission of disease and unstable supply of shortcomings. Using mammary gland bioreactor, making exogenous protein specific expressed in breast and secreted through breast milk, which has become an important means of producing secure and stable scarce protein. TALEN-mediated gene-editing technologies able to position insert foreign genes in the genome, it has many advantages compared to random integration, At the sime time, using transgenic cells, through somatic cell nuclear transfer is the conventional means of producing transgenic animals. Based on this, we carried out the following work:1.We used position-targeting goat genomic β-lactoglobulin first exon TALEN plasmid which preserved in the laboratory, and used this plasmid as the basis to build the corresponding dual marker activity HSA targeting vector pgB-HSA-puro(+);2. The tissue block method were carried on to culture the primary of goat mammary gland epithelial cells, using immunofluorescence staining of keratin 18 were used to identify mammary gland epithelial cells. Then targeting carrier pgB HSA-puro(+), TALEN plasmid were co-transfected into goat mammary epithelial cells, using a cell culture medium containing 1 000 ng/mL puro to select the positive clones, and through the hormone induced in positive mammary gland epithelial cells, endogenous BLG promoter is verified the validity of HSA expression regulation;3. We co-transfecting targeting vector pgB-HSA-puro(+), TALEN plasmid into goat fetal fibroblasts and using a cell culture medium containing 1 000 ng/mL puro to select the positive clones, which used as donor cells, the trans-HAS gene cloned blastocyst were obtained by somatic cell nuclear transfer.Finally, we successful constructed HSA targeting vector pgB-HSA-puro(+), which positioning targeting at first exon of goat β-lactoglobulin and screened TALEN mediated trans-HSA gene goat fibroblast cell clones, whoes targeting efficiency was 11%; the clones provides new cell materials for the study of the related genetically modified; Through somatic cell nuclear transfer, trans-HSA cloning blastocyst was obtained, the highest blastocyst rate was 20.8%. This study has verified the feasibility of the use of goat mammary gland bioreactor produce HAS protein and lay the foundation of the next step of trans-HAS gene goat,s production...