猪前脂肪细胞为核供体生产表达绿色荧光蛋白的转基因克隆胚胎

分离培养了成年猪前脂肪原代细胞，并对前脂肪细胞向成熟脂肪细胞进行诱导分化，油红O染色法鉴定了脂肪细胞。利用脂质体介导的方法将质粒pEGFP—N1转染前脂肪细胞，经过G418筛选获得了阳性细胞株。然后分别以前脂肪细胞、转绿色荧光蛋白（GFP）前脂肪细胞及胎儿成纤维细胞为核供体进行体细胞核移植，结果表明：前脂肪细胞（8．8％）和胎儿成纤维细胞（8．3％）的囊胚发育率无显差异著（P〉0．05）；与前脂肪细胞相比，转基因前脂肪细胞的囊胚发育率降低，但差异不显著（3．7％ vs．8．8％，P〉0．05）。前脂肪细胞为核供体生产转基因克隆胚胎，能够获得转基因囊胚。虽然囊胚发育率不高，但为生产脂肪细胞转基因克隆猪奠定了基础。

Production of Transgenic Embryos Expressing Green Fluorescent Protein by Nuclear Transfer of Preadipocytes in Pig

Primary culture of porcine preadipocytes was established using fat tissue from an adult pig.The preadipocytes to re-differentiate induction into mature adipocytes was confirmed by oil red O staining.A porcine preadipocyte(PreA) lines were transfected with pEGFP-N1 plasmid DNA by lipofectin-mediated way.GFP-PreA clones were obtained by G418 selection.PreA,fetal fibroblast(FF),and GFP-PreA were used as nuclear donors for somatic cell nuclear transfer in pig.The results were as follows: For the blastocyst rate of cloned embryos,no significant difference was observed between PreA and FF(8.8% vs 8.3%,P > 0.05).Blastocyst rate of reconstructed with GFP transfected PreA decreased in comparison to those derived from non-transgenic PreA.(3.7% vs.8.8%,P > 0.05).The results indicate that GFP transfected PreA can produce procine transgenic blastocytes.In spite of low blastocyst rate,cloning pigs from preadipocyte is possible.