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Quantifying In Vivo Somatic Mutations Using Transgenic Mouse Model Systems

  • Roy R. Swiger
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1105)

Abstract

This chapter describes the use of the bacteriophage cII positive selection somatic mutational assay with the Muta™Mouse transgenic model system. The assay is similar to others involving a transgenic target, including the cII and lacI assays in the Big Blue® Mouse, lacZ in the MutaMouse, and the gpt delta assay. Briefly, high-molecular-weight DNA is purified from the tissue of interest and used as substrate during in vitro packaging reactions, where the λ transgenes are excised from the genome and assembled into viable phage. Phage containing the mutational targets is then adsorbed into an appropriate bacterial host, and mutations sustained in vivo are detected and quantified by either standard recombinant screening or selection assays. Mutant frequencies are reported as the ratio of mutant phage to total phage units analyzed. The λ-based transgenic mouse assays are used to study and characterize in vivo mutagenesis as well as for mutagenicity assessment of chemicals and other agents. These models permit the enumeration of mutations sustained in virtually any tissue of the mouse and are both sensitive and robust. Application of the assays is simple, not requiring resources beyond those commonly found in most academic laboratories.

Key words

cII gpt lacZ lacI Mutation assay Genotoxicity Big Blue Induced MutaMouse Mutagen Mutation Somatic Spontaneous Tissue 

Notes

Acknowledgments

I wish to thank Professor John A. Heddle, York University, for introducing me to and educating me on the transgenic models. Thanks also to Ms. Lorien Newell for useful comments.

References

  1. 1.
    Leach EG, Narayanan L, Havre PA, Gunther EJ, Yeasky TM, Glazer PM (1996) Tissue specificity of spontaneous point mutations in lambda supF transgenic mice. Environ Mol Mutagen 28:459–464PubMedCrossRefGoogle Scholar
  2. 2.
    Gossen JA, de Leeuw WJF, Tan CHT et al (1989) Efficient rescue of integrated shuttle vectors from transgenic mice: a model for studying mutations in vivo. Proc Natl Acad Sci U S A 86:7971–7975PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Kohler SW, Provost GS, Fieck A et al (1991) Spectra of spontaneous and mutagen-induced mutations in the lacI gene in transgenic mice. Proc Natl Acad Sci U S A 88:7958–7962PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Nohmi T, Katoh M, Suzuki H et al (1996) A new transgenic mouse mutagenesis test system using Spi- and 6-thioguanine selections. Environ Mol Mutagen 28:465–470PubMedCrossRefGoogle Scholar
  5. 5.
    Douglas GR, Jiao J, Gingerich JD, Gossen JA, Soper LM (1995) Temporal and molecular characteristics of mutations induced by ethylnitrosourea in germ cells isolated from seminiferous tubules and in spermatozoa of lacZ transgenic mice. Proc Natl Acad Sci U S A 92:7485–7489PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Zhang XB, Urlando C, Tao KS, Heddle JA (1995) Factors affecting somatic mutation frequencies in vivo. Mutat Res 338:189–201PubMedCrossRefGoogle Scholar
  7. 7.
    Swiger RR, Cosentino L, Masumura KI, Nohmi T, Heddle JA (2001) Further characterization and validation of gpt delta transgenic mice for quantifying somatic mutations in vivo. Environ Mol Mutagen 37:297–303PubMedCrossRefGoogle Scholar
  8. 8.
    Tao KS, Urlando C, Heddle JA (1993) Comparison of somatic mutation in a transgenic versus host locus. Proc Natl Acad Sci U S A 90:10681–10685PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Walker VE, Gorelick NJ, Andrews JL et al (1996) Frequency and spectrum of ethylnitrosourea-induced mutation at the hprt and lacI loci in splenic lymphocytes of exposed lacI transgenic mice. Cancer Res 56: 4654–4661PubMedGoogle Scholar
  10. 10.
    Cosentino L, Heddle JA (1999) A comparison of the effects of diverse mutagens at the lacZ transgene and Dlb-1 locus in vivo. Mutagenesis 14:113–119PubMedCrossRefGoogle Scholar
  11. 11.
    Shaver-Walker PM, Urlando C, Tao KS, Zhang XB, Heddle JA (1995) Enhanced somatic mutation rates induced in stem cells of mice by low chronic exposure to ethylnitrosourea. Proc Natl Acad Sci U S A 92:11470–11474PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Skopek TR, Kort KL, Marino DR et al (1996) Mutagenic response of the endogenous hprt gene and lacI transgene in benzo[a]pyrene-treated Big Blue B6C3F1 mice. Environ Mol Mutagen 28:376–384PubMedCrossRefGoogle Scholar
  13. 13.
    Cosentino L, Heddle JA (2000) Differential mutation of transgenic and endogenous loci in vivo. Mutat Res 454:1–10PubMedCrossRefGoogle Scholar
  14. 14.
    Jakubczak JL, Merlino G, French JE et al (1996) Analysis of genetic instability during mammary tumor progression using a novel selection-based assay for in vivo mutations in a bacteriophage lambda transgene target. Proc Natl Acad Sci U S A 93:9073–9078PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Swiger RR, Cosentino L, Shima N, Bielas JH, Cruz-Munoz W, Heddle JA (1999) The cII locus in the Muta™Mouse system. Environ Mol Mutagen 34:201–207PubMedCrossRefGoogle Scholar
  16. 16.
    Winn NR, Norris MB, Brayer KJ, Torres C, Muller SL (2000) Detection of mutations in transgenic fish carrying a bacteriophage λcII transgene target. Proc Natl Acad Sci U S A 97:12655–12660PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Zimmer DM, Harbach PR, Mattes WB, Aaron CS (1999) Comparison of mutant frequencies at the transgenic lambda LacI and cII/cI loci in control and ENU-treated Big Blue mice. Environ Mol Mutagen 33:249–256PubMedCrossRefGoogle Scholar
  18. 18.
    Watson DE, Cunningham ML, Tindall KR (1998) Spontaneous and ENU-induced mutation spectra at the cII locus in Big Blue Rat2 embryonic fibroblasts. Mutagenesis 13: 487–497PubMedCrossRefGoogle Scholar
  19. 19.
    Harbach PR, Zimmer DM, Filipunas AL, Mattes WB, Aaron CS (1999) Spontaneous mutation spectrum at the lambda cII locus in liver, lung, and spleen tissue of Big Blue transgenic mice. Environ Mol Mutagen 33: 132–143PubMedCrossRefGoogle Scholar
  20. 20.
    You YH, Pfeifer GP (2001) Similarities in sunlight-induced mutational spectra of CpG-methylated transgenes and the p53 gene in skin cancer point to an important role of 5-methylcytosine residues in solar UV mutagenesis. J Mol Biol 305:389–399PubMedCrossRefGoogle Scholar
  21. 21.
    Shima N, Swiger RR, Heddle JA (2000) Dietary restriction during murine development provides protection against MNU-induced mutations. Mutat Res 470:189–200PubMedCrossRefGoogle Scholar
  22. 22.
    Suzuki T, Wang X, Miyata Y et al (2000) Hepatocarcinogen quinoline induces G:C to C:G transversions in the cII gene in the liver of lambda/lacZ transgenic mice (Muta™Mouse). Mutat Res 456:73–81PubMedCrossRefGoogle Scholar
  23. 23.
    Kohara A, Suzuki T, Honma M et al (2001) Mutation spectrum of o-aminoazotoluene in the cII gene of lambda/lacZ transgenic mice (Muta™Mouse). Mutat Res 491:211–220PubMedCrossRefGoogle Scholar
  24. 24.
    Morgan C, Lewis PD (2006) iMARS—mutation analysis reporting software: an analysis of spontaneous cII mutation spectra. Mutat Res 603:15–26PubMedCrossRefGoogle Scholar
  25. 25.
    Arit VM, Gingerich J, Schmeiser HH et al (2008) Genotoxicity of 3-nitrobenzaththrone in MutaMouse and lung epithelial cell derived from MutaMouse. Mutagenesis 23:483–490CrossRefGoogle Scholar
  26. 26.
    Chen G, Gingerich J, Soper L, Douglas GR, White PA (2010) Induction of lacZ mutations in MutaMouse primary hepatocytes. Environ Mol Mutagen 51:330–337PubMedCentralPubMedGoogle Scholar
  27. 27.
    Jacobsen NR, Møller P, Cohn CA et al (2008) Diesel exhaust particles are mutagenic in FE1-MutaMouse lung epithelial cells. Mutat Res 641:54–57PubMedCrossRefGoogle Scholar
  28. 28.
    Berndt-Weis ML, Kauri LM, Williams A et al (2009) Global transcriptional characterization of a mouse pulmonary epithelial cell line for use in genetic toxicology. Toxicol In Vitro 23:816–833PubMedCrossRefGoogle Scholar
  29. 29.
    Beyerle A, Long AS, White PA, Kissel T, Stoeger T (2011) Poly(ethylene imine) nanocarriers do not induce mutations nor oxidative DNA damage in vitro in MutaMouse FE1 cells. Mol Pharmacol 8:976–981CrossRefGoogle Scholar
  30. 30.
    Sambrook J, Fritsch EF, Manniatis T (eds) (1989) Molecular cloning: a laboratory manual, 2nd ed., vol 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  31. 31.
    Straus WM (1994) Preparation of genomic DNA from mammalian tissue. In: Ausubel FM, Brent R, Kingston RE et al (eds) Current protocols in molecular biology, vol 1, suppl 13 and 25. Wiley Interscience, New York, pp 221–223Google Scholar
  32. 32.
    Poustka A (1993) Construction and use of chromosome jumping libraries. Methods Enzymol 217:358–378PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press 2014

Authors and Affiliations

  1. 1.Florida DivisionMidwest Research InstitutePalm BayUSA

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