摘要
本研究旨在构建溶葡萄球菌素基因牛乳腺特异表达载体(pEPB),转染牛胎儿成纤维细胞,为制备溶葡萄球菌素基因的转基因克隆牛提供核供体细胞。本研究以pEGFP-C1为载体骨架,通过PCR扩增牛2.6kb的β-酪蛋白5′调控区及0.6kb的3′侧翼区(poly A)序列作为调控序列,制备成含有绿色荧光和新霉素筛选标记的牛乳腺特异表达载体,经PCR和酶切鉴定正确后,用转染试剂FuGene HD反复转染牛pEPB乳腺上皮细胞3～5次,用催乳素诱导后,经免疫荧光分析,检测目的蛋白的表达。然后,用电转染法转染牛胎儿成纤维细胞,经G418筛选得到阳性细胞后,把阳性细胞扩大培养并提取其基因组,因为溶葡萄球菌素基因是外源基因,经PCR及Southern blot检测来确定目的基因是否已经整合到细胞的基因组上。结果表明,溶葡萄球菌素基因在牛乳腺细胞中得到了表达,并整合到牛胎儿成纤维细胞的基因组中,得到了转溶葡萄球菌素基因的核供体细胞。结果显示,本研究所获得的转基因牛胎儿成纤维细胞可作为体细胞核移植的供体细胞进行转基因克隆牛研究。
The purpose of this study was to prepare lysostaphin transgenic donor cells for somatic cell nuclear transfer by constructing mammary-specific expression vector and transfecting bovine fetal fibroblasts.In this study,pEGFP-C1 was used as the vector backbone,2.6 kb 5′ regulatory region of and 0.6 kb 3′ flanking region of bovine β-casein were used as regulatory regions,the mammary gland-specific expression vector pEPB was constructed,which included neomycin resistant gene and EGFP reporter gene.After identified by PCR and restrictive enzyme digestion,the pEPB was transfected into the bovine mammary epithelial cells through FuGene HD for 3-5 times,the transgenic cells were detected by immunofluorescence analysis after induced by prolactin（1 mg·mL-1）.And then,the plasmid pEPB was transfected into the bovine fetal fibroblast cells by electroporation,positive cells were screened by G418 selection and determined by PCR and Southern blot.The results showed that the lysostaphin gene was expressed in bovine mammary gland epithelial cells and integrated into bovine fetal fibroblasts genome.The results indicate that the lysostaphin transgenic fibroblast cells obtained may be competent for bovine transgenic cloning.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2011年第10期1374-1379,共6页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家转基因生物新品种培育重大专项"抗病转基因新品种培育"(2008ZX08007)
