Generation of knockout mice using engineered nucleases

Young Hoon Sung, Young Jin, Seokjoong Kim, Han Woong Lee

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

The use of engineered nucleases in one-cell stage mouse embryos is emerging as an efficient alternative to conventional gene targeting in mouse embryonic stem (ES) cells. These nucleases are designed or reprogrammed to specifically induce double strand breaks (DSBs) at a desired genomic locus, and efficiently introduce mutations by both error-prone and error-free DNA repair mechanisms. Since these mutations frequently result in the loss or alteration of gene function by inserting, deleting, or substituting nucleotide sequences, engineered nucleases are enabling us to efficiently generate gene knockout and knockin mice. Three kinds of engineered endonucleases have been developed and successfully applied to the generation of mutant mice: zinc-finger nuclease (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided endonucleases (RGENs). Based on recent advances, here we provide experimentally validated, detailed guidelines for generating non-homologous end-joining (NHEJ)-mediated mutant mice by microinjecting TALENs and RGENs into the cytoplasm or the pronucleus of one-cell stage mouse embryos.

Original languageEnglish
Pages (from-to)85-93
Number of pages9
JournalMethods
Volume69
Issue number1
DOIs
Publication statusPublished - 2014 Jan 1

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

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