四倍体补偿技术的建立及其应用

常规基因剔除小鼠的获得主要是利用ES细胞的全能性先获得嵌合体小鼠,再利用:ES细胞的生殖系传递能力,通过嵌合体与野生型小鼠的交配获得杂合子小鼠.而四倍体补偿技术则可绕过嵌合体小鼠阶段,直接获得基因修饰杂合子小鼠.利用电融合技术和Piezoelectric microinjecfion显微注射技术建立了四倍体补偿技术,小鼠四倍体胚胎的获得率(电融合率)为(93.01±l.37)%,经体外培养囊胚形成率为(82.49±2.08)%.通过显微注射方法将2种129品系小鼠来源的ES细胞(CJ7和SCR012)注射到四倍体囊胚腔中,获得了完全ES细胞来源的小鼠,ES鼠的获得率分别为2.7%和8.3%.经微卫星DNA检测,成体小鼠的10个被检测组织均为129小鼠来源的.同时,也利用基因修饰的ES细胞进行了研究,获得了2种基因修饰的完全ES细胞来源的杂合子小鼠,部分小鼠具有繁殖能力,经繁育已获得了纯合子,其中凝血因子Ⅷ基因敲除小鼠获得了预期的血友病小鼠表型.上述结果说明四倍体补偿技术可应用于基因修饰小鼠的制备.

Production and Application of Mice Completely Derived from Inbred ES Cell Lines

The standard protocol for generating mutant mice from heterozygous targeted ES cells currently is time-consuming and needs two breeding steps and, in some cases, risks the failure of germline transmission. In contrast, the tetraploid embryo complementation is a time-saving single step procedure escaping from the chimera mouse. Hybrid ES cell lines always have higher generation frequency because of their genetic heterozygosities, which also limit their application. However, inbred ES cell lines often fail to generate ES mice. Judged by microsatellite DNA marker analysis, the completely ES cell derived mice (ES mice) were efficiently produced with two inbred ES cell lines (SCR012 and CJ7) by microinjecting the ES cells into tetraploid blastocysts generated by electrofusion. As a result, after electrofusion, about (93.01±1.37)% of 2-cell stage embryos fused and (82.49±2.08)% of them developed to blastocysts after 48 h in vitro culture. The efficiency of generating newborn ES mice of wild type SCR012 cells was higher (8.3%) than that of CJ7 (2.7%). In the meanwhile, adult and fertile mutant ES mice with two gene knock-out ES cells-18 KO (derived from CJ7) and F8 KO (derived from SCR012) were generated. Interestingly, the generation frequency of 18 KO ES mice was even higher (8.1%) than wild-type CJ7 ES mice, indicating that the development potential among ES cell clones was significantly different from each other and the decline of development potential was not equal among ES cell clones during ES cell passage in vitro culture. The F8 KO mice had indistinguishable bleeding phenotype, which is identical to that of mice derived from breeding of chimeric mice. These results suggested that this technique can be used as a powerful approach to produced gene targeted mice efficiently.