提高利用C57BL/6胚胎干细胞系制作基因打靶小鼠效率的研究  

Efficiency Improvement in Generation of Gene Targeted Mice with C57BL/6 Embryonic Stem Cells

作　　者：周舒雅[1] 左琴[1] 刘甦苏[1] 王辰飞 李保文[1] 贺争鸣[1] 范昌发[1] 

机构地区：[1]中国食品药品检定研究院实验动物资源研究所国家啮齿类实验动物种子中心,北京100050 [2]北京百奥赛图基因生物技术有限公司,北京101111

出　　处：《中国细胞生物学学报》2015年第6期780-786,共7页Chinese Journal of Cell Biology

基　　金：国家自然科学基金青年科学基金项目(批准号:81401661);中国食品药品检定研究院中青年发展基金课题(批准号:2012B5)资助的课题~~

摘　　要：为了提高通过C57BL/6（B6）胚胎干细胞（embryonic stem cells,ES cells）获得基因打靶小鼠的效率,该研究利用经过体外和体内多能性验证的C57BL/6 ES细胞系B6-1-6,开展了37组平行的制作基因打靶小鼠的实验。首先,对不同的囊胚获取的方式进行对比;其次,统计37组实验中的嵌合鼠表观嵌合度与种系遗传效率,并对二者相关性进行分析。对于BALB/c小鼠,自然排卵相较于激素超排能够得到更多囊胚（2.91个囊胚/只vs 0.82个囊胚/只）;对于最佳注射个数的探索,利用C57BL/6 ES细胞系B6-1-6进行基因修饰并注射囊胚,注射65-97个胚胎获得阳性打靶动物的可能性为95%;嵌合鼠毛色嵌合率与种系遗传率有一定相关性（r=0.316,P=0.057）,而嵌合鼠眼睛颜色与种系遗传效率显著正相关（r=0.328,P〈0.05）。该研究探索了用于C57BL/6 ES细胞注射的BALB/c囊胚获取方式的优化和最佳的注射数量,以及嵌合鼠表观嵌合度与种系遗传效率相关性等方面,以期为相关的研究提供有益的参考。Pluripotency of a C57BL/6 embryonic stem（ES） cell line, which was named as B6-1-6, was identified in vitro and in vivo. To improve efficiency of generating gene targeted mice, 37 different gene targeted mice were generated with B6-1-6. Different ways of inducing BALB/c mice ovulation were compared, and the correlation between the apparent chimerism and germline transmission efficiency was analyzed. Based on our data, we found that natural ovulation with estrus induction was superior to hormone superovulation for BALB/c mice（2.91 blasto./mouse vs 0.82 blasto./mouse）. For C57BL/6 ES cell line B6-1-6, 65~97 blastocysts should be injected to generate positive gene targeted mice with 95% success rate. In addition, we also found that the germline transmission efficiency was positively correlated with coat color chimerism level to a certain extent（r=0.316, P=0.057）; and it was positively correlated with eye color chimerism level significantly（r=0.328, P〈0.05）. In this study, we explored a superior way to obtain BALB/c blastocysts and optimum number for microinjection. What＇s more, the correlation between apparent chimerism level and germline transmission was discussed. Therefore, it will be helpful to improve the efficiency of generating gene targeted mice with C57BL/6 ES cells in future application.

关 键 词：C57BL/6胚胎干细胞 基因打靶 BALB/c囊胚 嵌合鼠表观嵌合度 种系遗传 

分 类 号：Q78[生物学—分子生物学]