利用卵胞浆精子注射_ICSI_技术生产转基因小鼠.pdfVIP

22 5 Vol. 22 No. 5 2006 9 Chinese Journal of Biotechnology September 2006 (ICSI) Transgenic Mice Produced by Intracytoplasmic Sperm Injection * 李 明, 严阿勇, 姚 慧, 安晓荣, 苟克勉 * LI Ming, YAN AYong, YAO Hui, AN XiaoRong and GOU KeMian , , 00094 StatedKey Laboratoriesf orAgrobiotechnologies, College of Biological Sciences, ChinaAgricultural University, Beijing 00094, China 在掌握小鼠ICSI 技术的基础上, 进行了ICSI 技术生产转基因小鼠的研究来自成年KM 小鼠附睾尾的精子, 使用 加抗冻剂的HEPESCZB 溶液, 在液氮中冻融 次后, 用于本实验解冻精子与DNA 混匀 min 后, 精子头被显微注射到 B6D2F 小鼠成熟卵母细胞质中精子头与pEGFPN 环状DNA 共注射生产的ICSI 受精卵, 在 CZB 溶液中培养至囊胚期时, 39. % (923) 的囊胚表达GFP 基因精子注射后6h, 直接移植 ICSI 受精卵后, 7 只妊娠受体 一共产仔30 只, 效率为 238% ( 3026) Southern blot 分析其中 6 只小鼠发现, 3 只( 88%) 转基因小鼠同时整合了GFP 和Neomycin 基因, 它们全部来自精 子和线性DNA 混合的实验组( 阳性效率为333% , 39) , 相反, 精子与环状质粒 DNA 共注射生产的7 只ICSI 后代中, 没有检测 到外源基因转基因小鼠整合的外源基因能够传递给它们后代结果说明, 利用ICSI 技术可以高效地生产转基因小鼠, 宿主 基因组可能更容易整合线性化的外源基因 ICSI, 囊胚, 小鼠, 转基因, Southern blot Q78 A 000306 ( 2006) Abstract In our previous study, normal and fertilemicewere successful produced from oocytes following intracytoplasmic sperm injection ( ICSI) . In the present study, the possibility of producing transgenic embryos and offspring with this procedure was evaluated. After freezingthawed once using HEPESCZB medium without cryoprotectants, the cauda sperm from KM fertilemale were exposed to the circular or linear pEGFPN DNA for min and then coinjected into metaphase II oocytes of B6D2F strain. When the zygotes with two pronuclei were cultured in CZB medium to day 35, 39. % ( 923) of them, derived from oocytes coinjected with sperm head and pEGFPN plasmid DNA, were expressed GFP protein.