| Mammary gland bioreactor research is one of the most active areas in biotechnology, which can produce highly bioactive proteins. It has the advantage of low cost and simple purification. The validity and reasonability of gland specific recombinant structure is the basis of successful generation of animal mammary gland bioreactors. As a result, it is significant to construct a rapid detection system to evaluate recombinant vectors, which can lead to the high efficiency of generation of mammary gland bioreactors. In transgenic animal production, the technique of somatic cell nuclear transfer has many advantages. The main advantage is that transgenic procedure can be operated in somatic cell in vitro, which leads to great improvement of productive efficiency and decrease of productive cost. Therefore, It has become a hotspot field to combine the technology of gene transfer into cell and somatic nuclear transfer to generate gland bioreactors. In our studies, green fluorescent protein as a reporter gene was applied in the field of the mammary gland bioreactors, involved the construct of double selectable markers vector, establishment and characterization of goat fetal fibroblasts, optimization of transfection efficiency of goat fetal fibroblasts mediated by liposome and establishment of transgenic cells, and establishment of goat mammary epithelial and expression of BLG-EGFP. Firstly, enhanced green fluorescent protein (EGFP) gene was fused with neomycin phosphotransferase. Thus, the recombinant vector contained two selectable markers, EGFP and separated by an internal ribosomal entry site (IRES) of the encephalomyocarditis virus, which permits the separate expression of two genes driven by one promoter. This double selectable markers vector provided the basis for its further application in transgenic selection and gene expression research work. Fibroblasts derived from 90-day old or so goat fetus were successfully cultured with a normal tissue culture method, named after GFF. The cultured cells were analyzed by their morphology, adherence efficiency, growth curve, colony forming efficiency, chromosome and cell cycle. Results demonstrated that these cultured cells were typical fibroblasts with normal morphology, the normal ratio of karyotype was 87.04﹪. The culture and characterization analysis of goat fibroblasts provided a ready supply of donor cells for goat somatic transgenic cloning . The double selectable markers vector pEGFP-IRESneo plasmid coated with liposome reagent , was transferred into the goat fetal fibroblasts (GFF) to validate pEGFP-IRESneo. Such parameters affecting transfection efficiency as cell confluency, DNA amount, the ratio of liposome and DNA, the morphology of vector and serum were analyzed, and transfection efficiency was optimized by flow cytometry(FCM). It was found that the highest efficiency was achieved with 0.75μg annular DNA plasmid, the ratio of liposome and DNA at 1:2 , 80﹪confluent cells, in the absence of serum , demonstrating by the transfection efficiency of (40.7±3.12)%. Under the optimized condition of transfection, after selecting by the final concentrate of 800μg/mL G418 for 14 days and 400μg/mLG418 for 7 days, 3 transgenic GFF clones were obtained. And these cell clones were positive by detection of PCR. In addition, a goat mammary epithelia (GME) cell line was established from goat beastings. The cultured cells were analyzed by their morphology, histochemical identification. The cells exhibited the distinctive morphologic characteristics of mammary epithelial cells and expressed the special cytoskeleton protein of epithelial cells, cytokeratin 18. It was found that these cells could secret beta-casein by RT-PCR and ELISA , which further validated the epithelia characteristics of cultured cells. The GME transfected by mammarygland-specific expression recombinant, BLG-EGFP, could express green fluorescent protein, under the inducement of hormones, which indicated construct of BLG-EGFP was reasonable. All these results provided the basis for mammary gland bioreactors.
|
PDF Full Text Request