Knockout血清替代品可提高C57BL/6J小鼠胚胎干细胞建系效率

Knockout serum replacement improves establishment efficiency of C57BL/6J mouse embryonic stem cell line

目的:在培养液中添加knockout血清替代品(knockout serum replacement,KSR)代替胎牛血清(FBS)用于建立C57BL/6J小鼠胚胎干细胞(ESC)细胞系,以便消除血清中的不确定因子对ESC增殖的影响。方法:以C57BL/6J小鼠3.5 d的囊胚为材料分离ESC,比较KSR和FBS用于建立小鼠ESC细胞系的效率,并通过体内、外分化验证所分离获得的小鼠ESC的发育潜能。结果:培养液中添加KSR,成功从13个小鼠囊胚中分离获得一个ESC细胞系(MES-1),体外培养传代超过20代仍保持未分化状态,核型为正常XX型,碱性磷酸酶及oct-4基因高表达,悬浮培养可以生成拟胚体,接种到裸鼠皮下可形成畸胎瘤,注射到ICR小鼠3.5 d囊胚中,ESC可以参与胚胎发育并产生嵌合体小鼠。而培养液中添加FBS的对照组未能获得超过3代的ESC细胞系。结论:在培养液中添加KSR代替FBS适合于C57BL/6J小鼠ESC的分离与培养,从而可避免实验前对所用血清的筛选。

AIM： To eliminate the influence of serum on self-renewal of embryonic stem cells （ESCs）, knockout serum replacement （KSR）, a defined formulation, was used to replace serum for the establishment of C57BL/6J mouse ESC line, METHODS： C57BL/6J mouse blastocysts collected at 3.5 days post coitum （d. p. c. ） were cultured in the medium supplemented with KSR. In control experiment, KSR was substituted by fetal bovine serum （FBS）. When ESC line was established, the morphology of ESCs, the expression of alkaline phosphatase and oct-4, and the kary- otype and differentiating ability of ESCs were analyzed. RESULTS： 13 blastocysts were cultured in the medium supplemented with KSR and one ESC line （MES-1） was established with a normal and stable XX karyotype after cultured for more than 20 passages, and then the high expression of alkaline phosphatase and oct-4 was detected. When cul- tured in suspension, MES-1 formed embryoid bodies. When inoculated subcutaneously into nude mice, MES-1 formed teratoma. After injected into ICR mouse blastocysts collected at 3.,5 d. p. c., MES-1 incorporated into the inner cell mass of the host blastocyst and contributed to the development of a chimera. In control experiment, no ESC lines were cultured for more than 3 passages. CONCLUSION： KSR can be efficiently used to isolate and culture C57BL/6J mouse ESCs, which can eliminate traditional prescreening of FBS suitable for isolation and culture of ESCs.