Direct analysis of brain phenotypes via neural blastocyst complementation.
Direct analysis of brain phenotypes via neural blastocyst complementation.
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DOI：
10.1038/s41596-020-0364-y
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发表时间：
2020-10
期刊：
影响因子：
14.8
通讯作者：
Schwer B
中科院分区：
文献类型：
--
作者：
Dai HQ;Liang Z;Chang AN;Chapdelaine-Williams AM;Alvarado B;Pollen AA;Alt FW;Schwer B
我们提供了一种通过神经胚泡互补(NBC)从小鼠胚胎干细胞(ESCs)体内生成前脑结构的方案。我们开发这一方案是为了研究特定前脑区域的发育和功能，包括大脑皮层和海马体。我们描述了一个完整的工作流程，从通过CRISPR/Cas9介导性编辑修改ESCs中给定的基因组位置的方法，到产生可直接分析的ESC重组前脑区的小鼠嵌合体。该程序首先通过CRISPR/Cas9对小鼠ESCs进行基因编辑，这一过程可以在大约4到8周内完成。我们提供了在大约两到三周内实现ESCs荧光标记的方案，这使得可以在通过NBC产生的嵌合体中追踪注射的ESC来源的供体细胞。一旦修饰的ESCs准备就绪，通过将ESCs注射到遗传编程的囊胚中，在大约三周内产生NBC嵌合体，这些囊胚随后被转移到假孕寄养细胞中。我们的活体脑器官发生平台是高效的，允许在短短三个月内在整个有机体的背景下对基因和其他基因组因素进行功能和系统分析。胚胎干细胞经过CRISPR/Cas9介导的编辑，然后通过神经胚泡互补用于重建小鼠嵌合体中的前脑区域。
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 about four to eight weeks. We provide protocols to achieve fluorescent labeling of ESCs in about two to three 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 about three weeks via injection of ESCs into genetically programmed blastocysts that are subsequently transferred into pseudopregnant fosters. Our in vivo brain organogenesis platform is efficient, allowing functional and systematic analysis of genes and other genomic factors in as little as three months, in the context of a whole organism. Embryonic stem cells undergo CRISPR/Cas9-mediated editing and are then used to reconstitute forebrain regions in mouse chimeras via neural blastocyst complementation.
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影响因子：
64.5
作者：
Wu J;Platero-Luengo A;Sakurai M;Sugawara A;Gil MA;Yamauchi T;Suzuki K;Bogliotti YS;Cuello C;Morales Valencia M;Okumura D;Luo J;Vilariño M;Parrilla I;Soto DA;Martinez CA;Hishida T;Sánchez-Bautista S;Martinez-Martinez ML;Wang H;Nohalez A;Aizawa E;Martinez-Redondo P;Ocampo A;Reddy P;Roca J;Maga EA;Esteban CR;Berggren WT;Nuñez Delicado E;Lajara J;Guillen I;Guillen P;Campistol JM;Martinez EA;Ross PJ;Izpisua Belmonte JC
通讯作者：
Izpisua Belmonte JC
影响因子：
9.2
作者：
CHEN, JZ;GORMAN, JR;ALT, FW
通讯作者：
ALT, FW
影响因子：
56.9
作者：
Lois, C;Hong, EJ;Baltimore, D
通讯作者：
Baltimore, D
影响因子：
14.8
作者：
Ran, F. Ann;Hsu, Patrick D.;Zhang, Feng
通讯作者：
Zhang, Feng
影响因子：
2.5
作者：
Ryder, Edward;Gleeson, Diane;Sethi, Debarati;Vyas, Sapna;Miklejewska, Evelina;Dalvi, Priya;Habib, Bishoy;Cook, Ross;Hardy, Matthew;Jhaveri, Kalpesh;Bottomley, Joanna;Wardle-Jones, Hannah;Bussell, James N.;Houghton, Richard;Salisbury, Jennifer;Skarnes, William C.;Ramirez-Solis, Ramiro
通讯作者：
Ramirez-Solis, Ramiro