Esmail Michael Y, Qi Peimin, Connor Aurora Burds, Fox James G, García Alexis
Division of Laboratory Animal Medicine, Tufts University, Boston, Massachusetts, USA.
Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
J Am Assoc Lab Anim Sci. 2016;55(4):400-5.
The reliable generation of high-percentage chimeras from gene-targeted C57BL/6 embryonic stem cells has proven challenging, despite optimization of cell culture and microinjection techniques. To improve the efficiency of this procedure, we compared the generation of chimeras by using 3 different inbred, albino host, embryo-generating protocols: BALB/cAnNTac (BALB/c) donor mice superovulated at 4 wk of age, 12-wk-old BALB/c donor mice without superovulation, and C57BL/6NTac-Tyr(tm1Arte) (albino B6) mice superovulated at 4 wk of age. Key parameters measured included the average number of injectable embryos per donor, the percentage of live pups born from the total number of embryos transferred to recipients, and the number of chimeric pups with high embryonic-stem-cell contribution by coat color. Although albino B6 donors produced significantly more injectable embryos than did BALB/c donors, 12-wk-old BALB/c donor produced high-percentage (at least 70%) chimeras more than 2.5 times as often as did albino B6 mice and 20 times more efficiently than did 4-wk-old BALB/c donors. These findings clearly suggest that 12-wk-old BALB/c mice be used as blastocyst donors to reduce the number of mice used to generate each chimera, reduce the production of low-percentage chimeras, and maximize the generation of high-percentage chimeras from C57BL/6 embryonic stem cells.
尽管对细胞培养和显微注射技术进行了优化，但从基因靶向的C57BL/6胚胎干细胞可靠地产生高比例嵌合体已被证明具有挑战性。为了提高这一过程的效率，我们比较了使用3种不同的近交系白化宿主胚胎生成方案产生嵌合体的情况：4周龄超排的BALB/cAnNTac（BALB/c）供体小鼠、12周龄未超排的BALB/c供体小鼠以及4周龄超排的C57BL/6NTac-Tyr(tm1Arte)（白化B6）小鼠。测量的关键参数包括每个供体可注射胚胎的平均数量、移植到受体的胚胎总数中出生的活仔百分比以及通过毛色判断的具有高胚胎干细胞贡献的嵌合仔数量。虽然白化B6供体产生的可注射胚胎明显多于BALB/c供体，但12周龄的BALB/c供体产生高比例（至少70%）嵌合体的频率是白化B6小鼠的2.5倍多，效率是4周龄BALB/c供体的20倍。这些发现清楚地表明，应使用12周龄的BALB/c小鼠作为囊胚供体，以减少用于产生每个嵌合体的小鼠数量，减少低比例嵌合体的产生，并最大限度地从C57BL/6胚胎干细胞中产生高比例嵌合体。
