title: Mouse genome engineering using designer nucleases.
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.
authors.affiliation: Institute of Laboratory Animal Science, University of Zurich.
authors.forename: Mario
authors.initials: M
authors.lastname: Hermann
authors.affiliation: Department of Genetics, Cell Biology & Development and Center for Genome Engineering, University of Minnesota.
authors.forename: Tomas
authors.initials: T
authors.lastname: Cermak
authors.affiliation: Department of Genetics, Cell Biology & Development and Center for Genome Engineering, University of Minnesota.
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Voytas
authors.affiliation: Institute of Laboratory Animal Science, University of Zurich; pawel.pelczar@ltk.uzh.ch.
authors.forename: Pawel
authors.initials: P
authors.lastname: Pelczar
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000502:physiology*; D059766:DNA End-Joining Repair; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005260:Female; D005818:Genetic Engineering / Q000379:methods*; D023281:Genomics / Q000379:methods*; D006801:Humans; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008845:Microinjections; D009865:Oocytes / Q000502:physiology; D012333:RNA, Messenger / Q000008:administration & dosage / Q000235:genetics; D011995:Recombination, Genetic
chemical_list: D012333:RNA, Messenger; D004720:Endonucleases
references.citation: Nat Biotechnol. 2013 Jan;31(1):23-4
references.pmid: 23302927
references.citation: Nucleic Acids Res. 2012 Apr;40(8):3741-52
references.pmid: 22169954
references.citation: Genesis. 2010 Apr;48(4):264-80
references.pmid: 20143345
references.citation: J Am Assoc Lab Anim Sci. 2011 Jul;50(4):471-8
references.pmid: 21838974
references.citation: J Mol Biol. 2010 Jul 2;400(1):96-107
references.pmid: 20447404
references.citation: Nat Biotechnol. 2013 Jan;31(1):76-81
references.pmid: 23242165
references.citation: Proc Natl Acad Sci U S A. 2013 Mar 5;110(10):3782-7
references.pmid: 23426636
references.citation: Nucleic Acids Res. 2012 Jul;40(Web Server issue):W117-22
references.pmid: 22693217
references.citation: J Biol Chem. 2012 Nov 9;287(46):38427-32
references.pmid: 23019344
references.citation: Theriogenology. 2006 Jun;65(9):1716-26
references.pmid: 16271754
references.citation: Nat Biotechnol. 2013 Mar;31(3):251-8
references.pmid: 23417094
references.citation: PLoS One. 2012;7(9):e41796
references.pmid: 22970113
references.citation: Nat Biotechnol. 2013 Sep;31(9):822-6
references.pmid: 23792628
references.citation: Science. 2013 Feb 15;339(6121):819-23
references.pmid: 23287718
references.citation: Nat Rev Genet. 2010 Sep;11(9):636-46
references.pmid: 20717154
references.citation: Nucleic Acids Res. 2011 Jul;39(12):e82
references.pmid: 21493687
references.citation: Nucleic Acids Res. 2012 Jul;40(12):5368-77
references.pmid: 22387464
references.citation: Nat Biotechnol. 2011 Feb;29(2):143-8
references.pmid: 21179091
references.citation: Cell. 2013 May 9;153(4):910-8
references.pmid: 23643243
references.citation: Nat Biotechnol. 2012 May;30(5):460-5
references.pmid: 22484455
references.citation: Genesis. 2010 Mar;48(3):151-60
references.pmid: 20095053
references.citation: Nat Protoc. 2012 Jan 05;7(1):171-92
references.pmid: 22222791
references.citation: Nat Biotechnol. 2013 Sep;31(9):827-32
references.pmid: 23873081
references.citation: Trends Biotechnol. 2013 Jul;31(7):397-405
references.pmid: 23664777
references.citation: Nat Methods. 2011 Jan;8(1):74-9
references.pmid: 21131970
references.citation: Nature. 2011 Jun 15;474(7351):337-42
references.pmid: 21677750
references.citation: Nat Rev Mol Cell Biol. 2013 Jan;14(1):49-55
references.pmid: 23169466
references.citation: Nat Biotechnol. 2011 Jan;29(1):64-7
references.pmid: 21151125
references.citation: Genetics. 2010 Oct;186(2):451-9
references.pmid: 20628038
references.citation: Proc Natl Acad Sci U S A. 2010 Aug 24;107(34):15022-6
references.pmid: 20686113
references.citation: Curr Protoc Mouse Biol. 2011 Mar 01;1(1):199-211
references.pmid: 26068993
references.citation: Proc Natl Acad Sci U S A. 2012 Jun 12;109(24):9354-9
references.pmid: 22660928
references.citation: Nat Rev Genet. 2005 Jun;6(6):507-12
references.pmid: 15931173
references.citation: Science. 2013 Feb 15;339(6121):823-6
references.pmid: 23287722
references.citation: BMC Bioinformatics. 2011 May 13;12:152
references.pmid: 21569489