| The gene knockout technology is a way of genetic modification with precise fixed-point. With the combination of gene targeting, animal transgenic and animal cloning technologies, there have been a series of theoretical and applied research of animal genomes modified, such as fixed-point gene repair, the treatment of genetic defects and inactivation of animal unfavorable gene. A large number of basic and applied research gave the basic support on improvement the expression rates of mammary gland bioreactor and reduce the rate of random integration, which provided important help to find ways to achieve targeted integration and mammary gland-specific expression. This will undoubtedly become hotspot and key point of animal genetic research in the future.(3-lactoglobulin (BLG) is one of the allergens in baby milk allergies. The elimination of allergen in milk is an important means to eliminate allergies, And it can improve the quality of dairy and dairy products. But the elimination methods cannot solute the BLG allergenicity problem fundamentally and thoroughly. To obtain one dairy goats breed with BLG gene knockout is a alternative method for resolving milk allergy, which also have good prospect in high quality milk production. The purpose of this stud is to construct the goat P-lactoglobulin gene knockout ear fibroblasts through stable transfection and double-drug selection with BLG knockout vector. And lay the foundation for production of new goats breeding, whose dairy don’t contain BLG.This study can be divided into two parts. First, the isolation and culture of fibroblasts using goat ear tissue culture method; second, construction of the goatβ-lactoglobulin gene knockout ear fibroblasts. The following main steps were carried out:(1) the goat ear tissue fibroblasts were isolated and cultured by tissue culture, and the fresh and frozen-thawed cells anabiosis, viability and growth curve were determined; (2) the targeting vectors pBLG2T were transfected into the goat ear fibroblasts by liposome transfection methods; (3) positive-negative drug selection with G418 and ganciclovir were carried up for a total of 17 days after transfection; (4) cell clones were screened for homologous recombination by PCR methods; (5) The karyotype of positive cells were determined and the cells viability were compared with the normal fibroblasts. The results are as follows:1. The goat ear fibroblasts were isolated and cultured successfully. The fresh was 97%, and the frozen-thawed cells survival rate was 92.25%. The cell growth curve was "S" type, displayed the lag phase, the log phase and stationary phase.2. The concentration of G418 was 400μg/mL for selection and 200p.g/mL for maintain. The cells were positive selected with G418 for 7d, negative selected with G418 and GANC for 10d, And a total of 12 cell clones were obtained at last.3. Cell clones that survive this double drug selection are then screened for homologous recombination by PCR, only number 1 cell clone had the neo gene strip, and the targeting strip, considered number 1 cell clone was homologous recombinant clone. The vitality of the positive cells was significantly less than that of the normal fibroblasts (1.556±0.026 vs 1.726±0.036, P <0.01). And the homologous recombinant clone had stability nuclear that could be used as donor cell. |
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