Laboratory for Cell Asymmetry, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan.
Division of Molecular and Medical Genetics, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
Methods Mol Biol. 2021;2312:309-320. doi: 10.1007/978-1-0716-1441-9_19.
Developments in genome-editing technology, especially CRISPR-Cas9, have revolutionized the way in which genetically engineered animals are generated. However, the process of generation includes microinjection to the one-cell stage embryo and the transfer of the microinjected embryo to the surrogate animals, which requires trained personnel. We recently reported the method includes introduction of CRISPR-Cas9 systems to the developing cerebral cortex via in utero electroporation thus generating gene-targeted neural stem cells in vivo. This technique is widely applicable for gene knockout, monitoring gene expression, and lineage analysis in developmental biology. In this chapter, the detailed protocol of EGFP (enhanced green fluorescent protein) knock-in method via in utero electroporation is described.
基因组编辑技术的发展，尤其是 CRISPR-Cas9 的发展，彻底改变了基因工程动物的产生方式。然而，产生过程包括将微注射到单细胞胚胎中，并将微注射的胚胎转移到代孕动物中，这需要经过训练的人员。我们最近报道了一种方法，通过在体电穿孔将 CRISPR-Cas9 系统引入发育中的大脑皮层，从而在体内产生基因靶向神经干细胞。这项技术在基因敲除、监测基因表达和发育生物学中的谱系分析中具有广泛的应用。在本章中，描述了通过在体电穿孔进行 EGFP（增强型绿色荧光蛋白）基因敲入的详细方案。
