Rat Genomics pp 151-167 | Cite as

ENU Mutagenesis to Generate Genetically Modified Rat Models

  • Ruben van Boxtel
  • Michael N. Gould
  • Edwin Cuppen
  • Bart M. G. Smits
Part of the Methods in Molecular Biology book series (MIMB, volume 597)


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.

Key words

ENU mutagenesis Rat strains Mutation discovery Mutation frequency Mutant Knockout models 


  1. 1.
    Capecchi MR (2005) Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet 6:507–512CrossRefPubMedGoogle Scholar
  2. 2.
    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 40:516–522CrossRefPubMedGoogle Scholar
  3. 3.
    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 76:5818–5819CrossRefPubMedGoogle Scholar
  4. 4.
    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 21:645–651CrossRefPubMedGoogle Scholar
  5. 5.
    Smits BM, Mudde J, Plasterk RH, Cuppen E (2004) Target-selected mutagenesis of the rat. Genomics 83:332–334CrossRefPubMedGoogle Scholar
  6. 6.
    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 40:514–515CrossRefPubMedGoogle Scholar
  7. 7.
    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 16:159–169PubMedGoogle Scholar
  8. 8.
    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 104:4036–4041CrossRefPubMedGoogle Scholar
  9. 9.
    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 26:1626–1635CrossRefPubMedGoogle Scholar
  10. 10.
    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 29:1290–1297CrossRefPubMedGoogle Scholar
  11. 11.
    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 146:1662–1676CrossRefPubMedGoogle Scholar
  12. 12.
    Chen KS, Gould MN (2004) Development of a universal gap repair vector for yeast-based screening of knockout rodents. Biotechniques 37:383–388PubMedGoogle Scholar
  13. 13.
    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 11:484–488CrossRefPubMedGoogle Scholar
  14. 14.
    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 63:2062–2066PubMedGoogle Scholar
  15. 15.
    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 9:460CrossRefPubMedGoogle Scholar
  16. 16.
    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 171–178Google Scholar
  17. 17.
    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 17:230–238CrossRefPubMedGoogle Scholar
  18. 18.
    Nickerson DA, Tobe VO, Taylor SL (1997) PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing. Nucleic Acids Res 25:2745–2751CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Ruben van Boxtel
    • 1
  • Michael N. Gould
    • 2
  • Edwin Cuppen
    • 3
  • Bart M. G. Smits
    • 2
  1. 1.Hubrect InstituteNiob KnawThe Netherlands
  2. 2.McArdle Lab for Cancer ResearchUniversity of Wisconsin-MadisonMadisonUSA
  3. 3.Hubrecht Institute, NIOB KNAWUtrechtThe Netherlands

Personalised recommendations