The system-level identification and analysis of molecular networks in mammals can be accelerated by 'next-generation' genetics, defined as genetics that does not require crossing of multiple generations of animals in order to achieve the desired genetic makeup. We have established a highly efficient procedure for producing knock-in (KI) mice within a single generation, by optimizing the genome-editing protocol for KI embryonic stem (ES) cells and the protocol for the generation of fully ES-cell-derived mice (ES mice). Using this protocol, the production of chimeric mice is eliminated, and, therefore, there is no requirement for the crossing of chimeric mice to produce mice that carry the KI gene in all cells of the body. Our procedure thus shortens the time required to produce KI ES mice from about a year to ∼3 months. Various kinds of KI ES mice can be produced with a minimized amount of work, facilitating the elucidation of organism-level phenomena using a systems biology approach. In this report, we describe the basic technologies and protocols for this procedure, and discuss the current challenges for next-generation mammalian genetics in organism-level systems biology studies.

中文翻译：

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从胚胎干细胞单代生产敲入小鼠。

哺乳动物的分子网络的系统级鉴定和分析可通过“下一代”遗传学来加速，“下一代”遗传学是指不需要杂交多代动物即可获得所需遗传组成的遗传学。我们已经通过优化KI胚胎干（ES）细胞的基因组编辑协议和用于产生完全ES细胞的小鼠的协议，建立了一种高效的程序，可在单代内生产敲入（KI）小鼠（ES小鼠）。使用此协议，消除了嵌合小鼠的生产，因此，不需要杂交嵌合小鼠来生产在人体所有细胞中都携带KI基因的小鼠。因此，我们的程序将生产KI ES小鼠所需的时间从大约一年缩短至大约3个月。可以用最少的工作量来生产各种KI ES小鼠，从而可以使用系统生物学方法简化生物水平现象的阐明。在本报告中，我们描述了此程序的基本技术和协议，并讨论了在生物体级系统生物学研究中下一代哺乳动物遗传学的当前挑战。