title: Generation of knockout mice using engineered nucleases.
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.
authors.affiliation: Department of Biochemistry, College of Life Science and Biotechnology, Laboratory Animal Research Center, Yonsei University, Seoul 120-749, Republic of Korea.
authors.forename: Young Hoon
authors.initials: YH
authors.lastname: Sung
authors.affiliation: Department of Biochemistry, College of Life Science and Biotechnology, Laboratory Animal Research Center, Yonsei University, Seoul 120-749, Republic of Korea.
authors.forename: Young
authors.initials: Y
authors.lastname: Jin
authors.affiliation: ToolGen, Inc., Byucksan Kyoungin Digital Valley 2-Cha, Geumcheon-Gu, Seoul 153-023, Republic of Korea.
authors.forename: Seokjoong
authors.initials: S
authors.lastname: Kim
authors.affiliation: Department of Biochemistry, College of Life Science and Biotechnology, Laboratory Animal Research Center, Yonsei University, Seoul 120-749, Republic of Korea. Electronic address: hwl@yonsei.ac.kr.
authors.forename: Han-Woong
authors.initials: HW
authors.lastname: Lee
mesh_terms: D000818:Animals; D053453:Caspase 9 / Q000235:genetics; D059766:DNA End-Joining Repair; D004622:Embryo, Mammalian; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics; D055786:Gene Knockout Techniques; D018345:Mice, Knockout / Q000235:genetics*; D008845:Microinjections
chemical_list: D004720:Endonucleases; D053453:Caspase 9
keywords: Engineered nuclease; Knockout mouse; Microinjection; NHEJ; RGEN; TALEN; ZFN