Protocol for De Novo Gene Targeting Via In Utero Electroporation

Methods Mol Biol. 2021:2312:309-320. doi: 10.1007/978-1-0716-1441-9_19.

Abstract

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.

Keywords: CRISPR-Cas9; De novo gene targeting; Developmental biology; In utero electroporation; Visualization of protein subcellular localization.

MeSH terms

  • Animals
  • Brain / embryology
  • Brain / metabolism*
  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Electroporation*
  • Female
  • Gene Expression Regulation, Developmental*
  • Gene Knock-In Techniques
  • Gene Targeting*
  • Gene Transfer Techniques*
  • Genes, Reporter
  • Gestational Age
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Mice
  • Mice, Inbred ICR
  • Pregnancy
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • RNA, Guide, CRISPR-Cas Systems / metabolism

Substances

  • RNA, Guide, CRISPR-Cas Systems
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • CRISPR-Associated Protein 9