Direct analysis of brain phenotypes via neural blastocyst complementation
Hai-Qiang Dai1, Zhuoyi Liang1, Amelia N Chang1
1Howard 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.
Nature Protocols
|August 12, 2020
Summary
This study presents a protocol for generating mouse forebrain structures using neural blastocyst complementation (NBC) with embryonic stem cells (ESCs). This method enables efficient in vivo analysis of brain development and gene function.
Area of Science:
- Developmental Biology
- Neuroscience
- Stem Cell Biology
Background:
- Studying forebrain development and function requires precise genetic manipulation and analysis.
- Existing methods may lack efficiency or the ability to analyze gene function in a whole-organism context.
Purpose of the Study:
- To provide a detailed protocol for generating specific forebrain structures in vivo using mouse embryonic stem cells (ESCs) via neural blastocyst complementation (NBC).
- To enable functional and systematic analysis of genes and genomic factors in the developing brain.
Main Methods:
- CRISPR-Cas9 gene editing of mouse ESCs.
- Fluorescent labeling of modified ESCs for cell tracing.
- Neural blastocyst complementation (NBC) by injecting ESCs into blastocysts.
- Generation and analysis of chimeric mice with ESC-reconstituted forebrain regions.
Main Results:
- A complete workflow from ESC genetic modification to chimera generation is described.
- The protocol allows for the generation of mouse chimeras with ESC-derived forebrain structures, including the cerebral cortex and hippocampus.
- The platform enables functional analysis of genes in vivo within 3 months.
Conclusions:
- This protocol offers an efficient in vivo platform for studying brain organogenesis and gene function.
- Neural blastocyst complementation (NBC) provides a powerful tool for developmental and functional neuroscience research.
- The method facilitates the direct analysis of gene function in specific forebrain regions within a whole organism.
