Mouse genome engineering using designer nucleases

Mario Hermann, Tomas Cermak, Daniel F Voytas, Pawel Pelczar

Research output: Contribution to journalArticle

13 Scopus citations

Abstract

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Original languageEnglish (US)
Article numbere50930
JournalJournal of Visualized Experiments
Issue number85
DOIs
StatePublished - Apr 2 2014

Keywords

  • Designer nucleases
  • Genetics
  • Genome engineering
  • Issue 86
  • Oocyte microinjection
  • TALEN
  • ZFN

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