Construction Of Diary Goat BLG Gene Knockout Vector

The natural BLG is one of the major milk allergens of cow and diary goat. The Elimination of the BLG milk allergen is an important method to eliminate the allergy and improve milk quality. The current methods had already obtained certain of effect on reducing the risk of suffering from BLG allergy, they couldn't radically solve the problem of BLG allergy. This study was to construct a dairy goat BLG replacement gene knockout vector, which were used to transfect goat fetal fibroblasts. In order to breeding the new types of dairy goat, the positive targeting cells, collected by antibiotics, were used as nuclear donors in nuclear transfer to produce BLG gene deletion goats, therefore this suplied a good method to solve the problem of milk allergy.The primers were designed according to the known sequence of BLG, then the homologous arms were amplified by PCR. The 5'homologous arm is 2264bp, the 3'homologous arm is 4461 bp. After that, the homologous arms were cloned into pMD18-T Simple vector and sequenced respectively, then the 5'homologous arm and 3'homologous arm were subconed into the gene targeting plasmid ploxpâ…¡successively, which contained the positive and negative selection gene. Subsequently, the gene knockout vector was identified by restriction endonuclease digestion and PCR. To verify the bioactivity of loxp locus, the constructed gene knockout vector was transformed into the BM25.8 expressing Cre recombinase. The result indicates that the diary goat BLG gene knockout vector pBLG2T with the deletion of exon2 was sucessfully constructed, and the positive neo gene can be deleted by Cre recombinase.In order to obtain BLG gene knock-out goat fetal fibroblasts(GFFs), female and male GFFs were transfected with the pBLG2T plasmid mediated by electroporation. After transfection, the GFFs were selected with positive-negative selection drugs(G418 and Ganc). 21 days later, positive cell clones were obtained. The neo gene was transferred into positive cell clones steadily by PCR. So this study laid a foundation to obtain transgenic goats by nuclear transfer.