Development of transfection technique of foreign gene using bovine ES-like cells as a vector

本研究旨在建立以牛胚胎干细胞或体细胞为载体的外源基因转染系统。1. 为了构建报告基因，将PGK启动子区域插入绿色荧光蛋白（GFP）基因的克隆位点，该基因具有自荧光性。2. 采用电穿孔或脂质转染的方法将GDP基因转染到牛乳腺细胞、耳细胞或成纤维细胞中。结果表明，转染GDP基因后，电穿孔法对乳腺细胞有效，脂肪转染法对牛成年耳细胞和胎儿成纤维细胞有效。3. 6头小牛从嵌合胚胎移植中出生，2头小牛在出生时或出生后一天死亡。微卫星DNA分析表明，肝脏细胞、小牛毛根细胞和胎盘细胞嵌合，来源于es样细胞和四倍体胚胎。4. 我们尝试用G418聚合仅8天的GFP阳性ES细胞，使二倍体或四倍体胚胎聚集。15 d时获得7个正常胚，其中1个胚在主要5个器官上均有GFP表达，4个胚在1个以上器官上均有GFP强烈表达。这些结果表明，G418短时间内转染的胚胎干细胞或体细胞能够用于植入前转基因胚胎和聚集嵌合体的选择。

The present was carried out to develop a new system for foreign gene transfer into bovine embryos using bovine embryonic stem (ES) cells or somatic cells as a vector. 1. In order to construct a reporter gene, PGK promoter region was inserted into a cloning site of green fluorescent protein (GFP) gene which show a self fluorescent. 2. Electroporation or lipofection methods were used to transfect GDP gene into bovine somatic cells as mammary cells, ear cells or fibroblast. It was shown that electroporation was effective for mammary cells and lipofection method was effective for bovine adult ear cells and fetal fibroblasts in transfection of GDP gene. 3. Six calves were born from the transfer of the chimeric embryos, two calves died at birth or a day after birth. The microsatellite DNA analysis showed that the cells of liver, roots of hair from calves and placenta proved to be chimeric and derived from ES-like cells and tetraploid embryos. 4. We attempted aggregation of diploid or tetraploid embryos with GFP positive ES cells which were selected for only 8 days with G418. At 15 days, 7 normal embryos were obtained, 1 out of 7 embryos showed GFP expression on main five organs, and 4 embryos were detected strongly GFP expression on more one organ under a stereotypic microscope. These results indicate that transfected ES cells or somatic cells selected in a short term with G418 were able to use enough for the selection of preimplantation transgenic embryos and for aggregation chimera.