Technology Evaluation and Development, The Jackson Laboratory, Bar Harbor, Maine, United States of America.
PLoS One. 2013 Jul 2;8(7):e67826. doi: 10.1371/journal.pone.0067826. Print 2013.
There is a continual need to improve efficiency in creating precise genetic modifications in mice using embryonic stem cells (ESCs). We describe a novel approach resulting in 100% germline transmission from competent injected ESCs. We developed an F1 mouse host embryo (Perfect Host, PH) that selectively ablates its own germ cells via tissue-specific induction of diphtheria toxin. This approach allows competent microinjected ESCs to fully dominate the germline, eliminating competition for this critical niche in the developing and adult animal. This is in contrast to conventional methods, where competition from host germ cells results in offspring derived from host cells and ESCs, necessitating extensive breeding of chimeras and genotyping to identify germline. The germline transmission process is also complicated by variability in the actual number of ESCs that colonize the germline niche and the proportion that are germline competent. To validate the PH approach we used ESC lines derived from 129 F1, BALB/cByJ, and BTBR backgrounds as well as an iPS line. Resulting chimeric males produced 194 offspring, all paternally derived from the introduced stem cells, with no offspring being derived from the host genome. We further tested this approach using eleven genetically modified C57BL/6N ESC lines (International Knockout Mouse Consortium). ESC germline transmission was observed in 9/11 (82%) lines using PH blastocysts, compared to 6/11 (55%) when conventional host blastocysts were used. Furthermore, less than 35% (83/240) of mice born in the first litters from conventional chimeras were confirmed to be of ESC-origin. By comparison, 100% (137/137) of the first litter offspring of PH chimeras were confirmed as ESC-derived. Together, these data demonstrate that the PH approach increases the probability of germline transmission and speeds the generation of ESC derived animals from chimeras. Collectively, this approach reduces the time and costs inherent in the production of genetically modified animals.
在利用胚胎干细胞（ESCs）精确创造遗传修饰的小鼠方面，不断需要提高效率。我们描述了一种新方法，可使 100%的生殖系传递来自有能力的注射 ESCs。我们开发了一种 F1 小鼠宿主胚胎（Perfect Host，PH），通过组织特异性诱导白喉毒素选择性地使自身生殖细胞失活。这种方法允许有能力的微注射 ESCs 完全主导生殖系，消除了在发育和成年动物中对这个关键生态位的竞争。这与传统方法形成对比，在传统方法中，宿主生殖细胞的竞争导致来自宿主细胞和 ESCs 的后代，需要对嵌合体进行广泛繁殖和基因分型以鉴定生殖系。生殖系传递过程也因定植生殖系生态位的实际 ESCs 数量以及具有生殖系能力的比例的可变性而变得复杂。为了验证 PH 方法，我们使用了源自 129F1、BALB/cByJ 和 BTBR 背景的 ESC 系以及 iPS 系。产生的嵌合雄性产生了 194 只后代，所有后代均来自引入的干细胞，没有来自宿主基因组的后代。我们使用 11 种遗传修饰的 C57BL/6N ESC 系（国际基因敲除小鼠联合会）进一步测试了这种方法。使用 PH 囊胚观察到 9/11（82%）线的 ESC 生殖系传递，而使用传统宿主囊胚时为 6/11（55%）。此外，在来自传统嵌合体的第一窝中出生的不到 35%（83/240）的小鼠被证实是 ESC 起源。相比之下，PH 嵌合体第一窝的 100%（137/137）后代被证实是 ESC 衍生的。这些数据共同表明，PH 方法增加了生殖系传递的可能性，并加快了嵌合体中 ESC 衍生动物的产生。总的来说，这种方法减少了生产遗传修饰动物所固有的时间和成本。
