Abstract
The rat is one of the most preferred model organisms in biomedical research and has been extremely useful for linking physiology and pathology to the genome. However, approaches to genetically modify specific genes in the rat germ line remain relatively scarce. To date, the most efficient approach for generating genetically modified rats has been the target-selected N-ethyl-N-nitrosourea (ENU) mutagenesis-based technology. Here, we describe the detailed protocols for ENU mutagenesis and mutant retrieval in the rat model organism.
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References
Capecchi MR (2005) Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet 6:507–512
Aitman TJ, Critser JK, Cuppen E, Dominiczak A, Fernandez-Suarez XM, Flint J, Gauguier D, Geurts AM, Gould M, Harris PC, Holmdahl R, Hubner N, Izsvak Z, Jacob HJ, Kuramoto T, Kwitek AE, Marrone A, Mashimo T, Moreno C, Mullins J, Mullins L, Olsson T, Pravenec M, Riley L, Saar K, Serikawa T, Shull JD, Szpirer C, Twigger SN, Voigt B, Worley K (2008) Progress and prospects in rat genetics: a community view. Nat Genet 40:516–522
Russell WL, Kelly EM, Hunsicker PR, Bangham JW, Maddux SC, Phipps EL (1979) Specific-locus test shows ethylnitrosourea to be the most potent mutagen in the mouse. Proc Natl Acad Sci U S A 76:5818–5819
Zan Y, Haag JD, Chen KS, Shepel LA, Wigington D, Wang YR, Hu R, Lopez-Guajardo CC, Brose HL, Porter KI, Leonard RA, Hitt AA, Schommer SL, Elegbede AF, Gould MN (2003) Production of knockout rats using ENU mutagenesis and a yeast-based screening assay. Nat Biotechnol 21:645–651
Smits BM, Mudde J, Plasterk RH, Cuppen E (2004) Target-selected mutagenesis of the rat. Genomics 83:332–334
Mashimo T, Yanagihara K, Tokuda S, Voigt B, Takizawa A, Nakajima R, Kato M, Hirabayashi M, Kuramoto T, Serikawa T (2008) An ENU-induced mutant archive for gene targeting in rats. Nat Genet 40:514–515
Smits BM, Mudde JB, van de Belt J, Verheul M, Olivier J, Homberg J, Guryev V, Cools AR, Ellenbroek BA, Plasterk RH, Cuppen E (2006) Generation of gene knockouts and mutant models in the laboratory rat by ENU-driven target-selected mutagenesis. Pharmacogenet Genomics 16:159–169
Amos-Landgraf JM, Kwong LN, Kendziorski CM, Reichelderfer M, Torrealba J, Weichert J, Haag JD, Chen KS, Waller JL, Gould MN, Dove WF (2007) A target-selected Apc-mutant rat kindred enhances the modeling of familial human colon cancer. Proc Natl Acad Sci U S A 104:4036–4041
Cotroneo MS, Haag JD, Zan Y, Lopez CC, Thuwajit P, Petukhova GV, Camerini-Otero RD, Gendron-Fitzpatrick A, Griep AE, Murphy CJ, Dubielzig RR, Gould MN (2007) Characterizing a rat Brca2 knockout model. Oncogene 26:1626–1635
van Boxtel R, Toonen PW, van Roekel HS, Verheul M, Smits BM, Korving J, de Bruin A, Cuppen E (2008) Lack of DNA mismatch repair protein MSH6 in the rat results in hereditary non-polyposis colorectal cancer-like tumorigenesis. Carcinogenesis 29:1290–1297
Homberg JR, Olivier JD, Smits BM, Mul JD, Mudde J, Verheul M, Nieuwenhuizen OF, Cools AR, Ronken E, Cremers T, Schoffelmeer AN, Ellenbroek BA, Cuppen E (2007) Characterization of the serotonin transporter knockout rat: a selective change in the functioning of the serotonergic system. Neuroscience 146:1662–1676
Chen KS, Gould MN (2004) Development of a universal gap repair vector for yeast-based screening of knockout rodents. Biotechniques 37:383–388
Justice MJ, Carpenter DA, Favor J, Neuhauser-Klaus A, Hrabe de Angelis M, Soewarto D, Moser A, Cordes S, Miller D, Chapman V, Weber JS, Rinchik EM, Hunsicker PR, Russell WL, Bode VC (2000) Effects of ENU dosage on mouse strains. Mamm Genome 11:484–488
Claij N, van der Wal A, Dekker M, Jansen L, Te Riele H (2003) DNA mismatch repair deficiency stimulates N-ethyl-N-nitrosourea-induced mutagenesis and lymphomagenesis. Cancer Res 63:2062–2066
van Boxtel R, Toonen PW, Verheul M, van Roekel HS, Nijman IJ, Guryev V, Cuppen E (2008) Improved generation of rat gene knockouts by target-selected mutagenesis in mismatch repair-deficient animals. BMC Genomics 9:460
Smits BM, Haag JD, Cuppen E, Gould MN (2007) Rat knockout and mutant models. In: Conn PM (ed) Sourcebook of models for biomedical research. Humana, Totowa, NJ, pp 171–178
Keays DA, Clark TG, Flint J (2006) Estimating the number of coding mutations in genotypic- and phenotypic-driven N-ethyl-N-nitrosourea (ENU) screens. Mamm Genome 17:230–238
Nickerson DA, Tobe VO, Taylor SL (1997) PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing. Nucleic Acids Res 25:2745–2751
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van Boxtel, R., Gould, M.N., Cuppen, E., Smits, B.M.G. (2010). ENU Mutagenesis to Generate Genetically Modified Rat Models. In: Anegon, I. (eds) Rat Genomics. Methods in Molecular Biology, vol 597. Humana Press. https://doi.org/10.1007/978-1-60327-389-3_11
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DOI: https://doi.org/10.1007/978-1-60327-389-3_11
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