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Direct analysis of brain phenotypes via neural blastocyst complementation.
Nature Protocols ( IF 13.1 ) Pub Date : 2020-08-10 , DOI: 10.1038/s41596-020-0364-y
Hai-Qiang Dai 1 , Zhuoyi Liang 1 , Amelia N Chang 1 , Aimee M Chapdelaine-Williams 1 , Beatriz Alvarado 2 , Alex A Pollen 2, 3 , Frederick W Alt 1 , Bjoern Schwer 2, 4
Affiliation  

We provide a protocol for generating forebrain structures in vivo from mouse embryonic stem cells (ESCs) via neural blastocyst complementation (NBC). We developed this protocol for studies of development and function of specific forebrain regions, including the cerebral cortex and hippocampus. We describe a complete workflow, from methods for modifying a given genomic locus in ESCs via CRISPR–Cas9-mediated editing to the generation of mouse chimeras with ESC-reconstituted forebrain regions that can be directly analyzed. The procedure begins with genetic editing of mouse ESCs via CRISPR–Cas9, which can be accomplished in ~4–8 weeks. We provide protocols to achieve fluorescent labeling of ESCs in ~2–3 weeks, which allows tracing of the injected, ESC-derived donor cells in chimeras generated via NBC. Once modified ESCs are ready, NBC chimeras are generated in ~3 weeks via injection of ESCs into genetically programmed blastocysts that are subsequently transferred into pseudo-pregnant fosters. Our in vivo brain organogenesis platform is efficient, allowing functional and systematic analysis of genes and other genomic factors in as little as 3 months, in the context of a whole organism.



中文翻译:


通过神经囊胚互补直接分析大脑表型。



我们提供了一种通过神经囊胚互补(NBC)从小鼠胚胎干细胞(ESC)体内生成前脑结构的方案。我们开发了该协议用于研究特定前脑区域(包括大脑皮层和海马体)的发育和功能。我们描述了一个完整的工作流程,从通过 CRISPR-Cas9 介导的编辑修改 ESC 中给定基因组位点的方法,到生成具有可直接分析的 ESC 重建前脑区域的小鼠嵌合体。该过程首先通过 CRISPR-Cas9 对小鼠 ESC 进行基因编辑,可在约 4-8 周内完成。我们提供在约 2-3 周内实现 ESC 荧光标记的方案,从而可以在通过 NBC 生成的嵌合体中追踪注射的 ESC 衍生供体细胞。一旦修饰的 ESC 准备就绪,通过将 ESC 注射到基因编程的囊胚中,NBC 嵌合体将在约 3 周内生成,随后将囊胚转移到假妊娠培养物中。我们的体内脑器官发生平台非常高效,可以在短短 3 个月内对整个有机体进行基因和其他基因组因素的功能和系统分析。

更新日期:2020-08-10
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