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Direct analysis of brain phenotypes via neural blastocyst complementation.
Dai, Hai-Qiang; Liang, Zhuoyi; Chang, Amelia N; Chapdelaine-Williams, Aimee M; Alvarado, Beatriz; Pollen, Alex A; Alt, Frederick W; Schwer, Bjoern.
Afiliación
  • Dai HQ; Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, Department of Genetics, and Department of Pediatrics, Harvard Medical School, Boston, MA, USA.
  • Liang Z; Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, Department of Genetics, and Department of Pediatrics, Harvard Medical School, Boston, MA, USA.
  • Chang AN; Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, Department of Genetics, and Department of Pediatrics, Harvard Medical School, Boston, MA, USA.
  • Chapdelaine-Williams AM; Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, Department of Genetics, and Department of Pediatrics, Harvard Medical School, Boston, MA, USA.
  • Alvarado B; Developmental and Stem Cell Biology Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
  • Pollen AA; Developmental and Stem Cell Biology Graduate Program, University of California, San Francisco, San Francisco, CA, USA.
  • Alt FW; Department of Neurology, The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Kavli Institute for Fundamental Neuroscience, University of California, San Francisco, San Francisco, CA, USA.
  • Schwer B; Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children's Hospital, Department of Genetics, and Department of Pediatrics, Harvard Medical School, Boston, MA, USA. alt@enders.tch.harvard.edu.
Nat Protoc ; 15(10): 3154-3181, 2020 10.
Article en En | MEDLINE | ID: mdl-32778838
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.
Asunto(s)

Texto completo: 1 Base de datos: MEDLINE Asunto principal: Encéfalo / Mapeo Encefálico / Células Madre Embrionarias de Ratones Idioma: En Revista: Nat Protoc Año: 2020 Tipo del documento: Article

Texto completo: 1 Base de datos: MEDLINE Asunto principal: Encéfalo / Mapeo Encefálico / Células Madre Embrionarias de Ratones Idioma: En Revista: Nat Protoc Año: 2020 Tipo del documento: Article