Mouse N‐ethyl‐N‐nitrosourea (ENU) Mutagenesis

Abstract

Mutagenesis is a powerful tool for understanding gene function and disease that has been used in many organisms from bacteria to fish. The chemical N‐ethyl‐N‐nitrosourea is being used as a supermutagen in mice to allow forward genetic screens. The addition of a complex mammal such as the mouse to the list of model organisms that have been analyzed by large‐scale mutagenesis will provide new mammalian models of human diseases and will allow an analysis of conserved and divergent evolution among species.

Keywords: mouse genetics; ENU mutagenesis; allelic series; gene function; balancer chromosome

Figure 1.

Recessive pedigree scheme for genome‐wide mutations. A male mouse is treated with ENU, which induces many DNA lesions in the spermatogonial stem cells of the testis. After mitosis and meiosis, the testis will become populated with clones of mutagenized sperm. Thus, the testis represents a mosaic of different ENU‐induced mutations (schematically indicated as dots; top left). New mutations will be transmitted to the first‐generation offspring and become fixed in the germ line. These mice become founders of a pedigree to test for new recessive mutations (solid gray box; m). Males are mated to a wild‐type female (open circle) to generate offspring that may carry the new mutation (solid gray) or may be wild type (open shapes). Without genetic markers, the genotype of the animal is not known (m?), since the mutation is recessive. Therefore, multiple daughters are mated back to the first‐generation founder father to generate offspring that may carry a new mutation. A pedigree is normally flagged as a mutant if more than one third‐generation offspring exhibits the same phenotype.

Figure 2.

Recessive pedigree scheme using a balancer chromosome. The balancer chromosome used in this pedigree is a Cre–loxP engineered inversion on mouse Chr 4. The end points of this inversion are about 30 cM apart and effectively suppress the recovery of viable recombinant products over the interval. One end of the inversion is tagged with the tyrosinase minigene (Tyr), which confers a dark pigment to an otherwise white coat. The other end is tagged with the K14‐agouti minigene, which confers a yellowish pigment to a pigmented mouse coat. Mice homozygous for the balancer are a dark chocolate color (Bal/Bal). When crossed with a Tyr −/− (white) mouse, the offspring that are heterozygous for the balancer are a grayish color (e.g. m/Bal). Male C57BL/6Brd Tyr −/− mice are mutagenized with ENU to generate many different mutations in their germ line (two representative germ‐line mutations are shown as blue and red mosaics). When the ENU‐treated males are mated to females that are homozygous for the balancer (shaded in black), the resulting first‐generation offspring will all be heterozygous for the balancer chromosome and will possibly inherit an ENU‐induced mutation from the male. If the new mutation lies on Chr 4, it is balanced with the coat color marked inversion, which will suppress recombination and allow for selection based on coat color in subsequent generations. ENU‐induced mutations that are not on Chr 4 will also be transmitted but will segregate randomly from the coat color. First‐generation males and females are then mated to mice homozygous for the balancer, resulting in two types of offspring: chocolate‐colored animals that do not carry a new mutation on Chr 4 (shaded black) or grayish animals that are heterozygous for the balancer and potentially a new ENU‐induced mutation on Chr 4 (shaded black and red or blue). Heterozygous offspring from this second generation are brother–sister mated to screen for new recessive mutations carried in their germ line. Mice carrying potential recessive mutations on Chr 4 (m/m) can be recognized by their white coat color and can be screened for clinically relevant phenotypes. If no white animals are produced in the third generation, it indicates the presence of an autosomal recessive lethal mutation. These lethal mutations can be easily maintained by brother–sister mating the heterozygous gray mice (m/Bal). Any chocolate‐colored animal (Bal/Bal) does not carry an ENU‐induced mutation on Chr 4; however, a new genome‐wide mutation may be observed in this population and may be further characterized using the mating scheme diagrammed in Figure . The incorporation of the coat color tag allows for the selection of mutations localized on a single chromosome, which are mapped upon their isolation by linkage to the coat color marker, while it also allows for recovery of mutations that segregate geneome‐wide.

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References

Coghill EL, Hugill A, Parkinson N, et al. (2002) A gene‐driven approach to the identification of ENU mutants in the mouse. Nature Genetics 30: 255–256.

Colvin JS, Bohne BA, Harding GW, McEwen DG and Ornitz DM (1996) Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3. Nature Genetics 12: 390–397.

Finkel E (2000) Australian ‘Ranch’ gears up to mass‐produce mutant mice. Science 288: 1572–1573.

Herron BJ, Lu W, Rao C, et al. (2002) Efficient generation and mapping of recessive developmental mutations using ENU mutagenesis. Nature Genetics 30: 185–189.

Hitotsumachi S, Carpenter DA and Russell WL (1985) Dose‐repetition increases the mutagenic effectiveness of N‐ethyl‐N‐nitrosourea in mouse spermatogonia. Proceedings of the National Academy of Sciences of the United States of America 82: 6619–6621.

Hrabe de Angelis MH, Flaswinkel H, Ruchs H, et al. (2000) Genome wide large scale production of mutant mice by ENU mutagenesis. Nature Genetics 25: 444–447.

Karim FD, Chang HC, Therrien M, et al. (1996) A screen for genes that function downstream of Ras1 during Drosophila eye development. Genetics 143: 315–329.

Kasarskis A, Manova K and Anderson KV (1998) A phenotype‐based screen for embryonic lethal mutations in the mouse. Proceedings of the National Academy of Sciences of the United States of America 95: 7485–7490.

King DP, Zhao Y, Sangoram AM, et al. (1997) Positional cloning of the mouse circadian Clock gene. Cell 89: 641–653.

Li C, Chen L, Iwata T, et al. (1999) A Lys644Glu substitution in fibroblast growth factor receptor 3 (FGFR3) causes dwarfism in mice by activation of STATs and ink4 cell cycle inhibitors. Human Molecular Genetics 8: 35–44.

Nolan PM, Peters J, Strivens M, et al. (2000) A systematic genome‐wide phenotype‐driven mutagenesis programme for gene function studies in the mouse. Nature Genetics 25: 440–443.

Peters J, Andrews SJ, Loutit JF and Glegg JB (1985) A mouse b‐globin mutant that is an exact model of hemoglobin Rainier in man. Genetics 110: 709–721.

Schumacher A, Faust C and Magnuson T (1996) Positional cloning of a global regulator of anterior–posterior patterning in mice. Nature 383: 250–253.

Thaung C, West K, Clark BJ, et al. (2002) ENU‐induced eye mutations in the mouse: models for human eye disease. Human Molecular Genetics 11: 755–767.

Tsai T‐F, Chen K‐S, Weber JS, Justice MJ and Beaudet AL (2002) Evidence for translational regulation of the Snurf‐Snrpn locus is compatible with a quantitative hypervariability model for genomic imprinting. Human Molecular Genetics 11: 1659–1668.

Further Reading

Beaudet AL and Jiang YJ (2002) A rheostat model for a rapid and reversible form of imprinting‐dependent evolution. American Journal of Human Genetics 70: 1389–1397.

Chen Y, Yee D, Dains K, et al. (2000) Genotype‐based screen for ENU‐induced mutations in mouse embryonic stem cells. Nature Genetics 24: 314–317.

Davis AP and Justice MJ (1998) Mouse alleles: if you've seen one, you haven't seen them all. Trends in Genetics 14: 438–441.

Justice MJ (1999) Mutagenesis of the mouse germline. In: Jackson I and Abbott C (eds.) Mouse Genetics and Transgenics: A Practical Approach, pp. 185–215. Oxford, UK: Oxford University Press.

Justice MJ, Noveroske JN, Weber JS, Zheng B and Bradley A (1999) Mouse ENU mutagenesis. Human Molecular Genetics 8: 1955–1963.

Munro RJ, Bergstrom RA, Zheng QY, et al. (2000) Mouse mutants from chemically mutagenized embryonic stem cells. Nature Genetics 24: 318–321.

Nadeau JH, Balling R, Barsh G, et al. (2001) Sequence interpretation. Functional annotation of mouse genome sequences. Science 291: 5507, 1251–1255.

Newhaus IM and Beier DR (1998) Efficient localization of mutations by interval haplotype analysis. Mammalian Genome 9: 150–154.

Nusslein‐Volhard C, Frohnhofer HG and Lehmann R (1987) Determination of anteroposterior polarity in Drosophila. Science 238: 1675–1681.

Zheng B, Sage M, Cai WW, et al. (1999) Engineering a balancer chromosome in the mouse. Nature Genetics 22: 375–378.

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Justice, Monica J, and Northrop, Jennifer L(Sep 2006) Mouse N‐ethyl‐N‐nitrosourea (ENU) Mutagenesis. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0006229]