Deconstructing Gene Function through ENU Mutagenesis

Abstract

A great majority of genes present in the human genome are also present in the mouse, thus making it an attractive mammalian model organism to study gene function and dysfunction. Over the past few decades, the ability to manipulate the mouse genome has been developed in a variety of ways. A complementary methodology to create mutations in the mouse is to use chemical mutagenesis. N‐ethyl‐N‐Nitrosourea (ENU) is the mutagen of choice for creating random point mutations model organisms. Advances in sequencing technologies have resulted in a rapid identification of the causative mutation. ENU mutagenesis is a powerful hypothesis‐generating approach to create new mouse models through both forward and reverse genetics approaches. Furthermore, the addition of challenges can identify mutations affecting specific pathways, and specific mutant lines or strains can be used to identify modifiers.

Key Concepts

  • ENU produces mainly point mutations randomly throughout the genome.
  • ENU mutagenesis can be used in both forward and reverse genetics approaches.
  • ENU phenotype‐driven screens do not require previous knowledge of the gene to create new mouse models or reveal gene function.
  • ENU phenotype‐driven screens can assign functions to specific protein domains.
  • ENU phenotype‐driven screens identify new mouse models by means of phenotyping.
  • High‐throughput sequencing technologies mean that the identification of the mutation underlying an observed phenotype is very rapid.
  • Phenotype‐driven screens can be applied along with specific challenges or mutations to reveal mutations affecting specific pathways or which modify existing phenotypes.

Keywords: ENU; ENU mutagenesis; mouse; mouse models; phenotype; genetics; modifiers; next‐generation sequencing

Figure 1. ENU mutations. ENU‐treated male mice produce sperm carrying unique arrays of ENU‐induced mutations. They are mated to nontreated wild‐type females producing G1 mice, each carrying a unique set of mutations. Note that as each G1 male is unique, intercrossing G1s will not produce mutations in homozygosis as each G1 carries different mutations.
Figure 2. Dominant and recessive screens breeding schemes. G0 – male mice treated with ENU are crossed to wild‐type females. G1 – each G1 mouse carries a unique array of heterozygote ENU mutations. Dominant mutations causing phenotypes can be identified. G2 – to identify recessive mutations, a unique G1 is crossed to wild‐type females of the same inbred strain (C3H) as the female used in the previous generation. All G2 mice carry mutations from the selected G1 father. G2 females are then backcrossed to their G1 father to produce G3s, where on average one in eight individuals will carry any given mutation in homozygosis. Using this breeding scheme, no homozygote mutations could be generated on the X chromosome, as the G1 male founder X chromosome must come from its wild‐type mother. Note that other recessive breeding schemes such as intercrossing G2s can also be employed. Although B6 and C3H are selected for this breeding scheme, any other inbred strains can be potentially used.
Figure 3. Phenotype‐ and gene‐driven screens. Both screens share the same root: an ENU‐mutagenised G0 male crossed with a nonmutagenised female producing G1 progeny. Phenotypic screens start with the finding of an interesting phenotype following the identification of the causative mutation. Gene‐driven screens start with a gene of interest, screening sperm and DNA archives of G1 ENU‐mutagenised males. Once mutations are identified, the stored sperm is used to produce mice that are then tested for phenotypes.
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References

Andrews TD, Whittle B and Field MA (2012) Massively parallel sequencing of the mouse exome to accurately identify rare, induced mutations: an immediate source for thousands of new mouse models. Open Biology 2: 120061–120076.

Arnold CN, Xia Y and Lin P (2011) Rapid identification of a disease allele in mouse through whole genome sequencing and bulk segregation analysis. Genetics 187: 633–641.

Arnold CN, Barnes MJ and Berger M (2012) ENU‐induced phenovariance in mice: inferences from 587 mutations. BMC Research Notes 5: 577–591.

Barbaric I, Wells S, Russ A and Dear TN (2007) Spectrum of ENU‐induced mutations in phenotype‐driven and gene‐driven screens in the mouse. Environmental and Molecular Mutagenesis 48: 124–142.

Beck JA, Lloyd S, Hafezparast M, et al. (2000) Genealogies of mouse inbred strains. Nature Genetics 24: 23–25.

Buchovecky CM, Turley SD, Brown HM, et al. (2013) A suppressor screen in Mecp2 mutant mice implicates cholesterol metabolism in Rett syndrome. Nature Genetics 45: 1013–1020.

Bull KR, Rimmer AJ, Siggs OM, et al. (2013) Unlocking the bottleneck in forward genetics using whole‐genome sequencing and identity by descent to isolate causative mutations. PLoS Genetics 9: e1003219.

De Stasio EA and Dorman S (2001) Optimization of ENU mutagenesis of Caenorhabditis elegans. Mutation Research 495 (1–2): 81–88.

Dickinson ME, Flenniken AM, Ji X, et al. (2016) High‐throughput discovery of novel developmental phenotypes. Nature 537: 508–514.

Gallego‐Llamas J, Timms AE, Pitstick R, et al. (2016) Improvement of ENU mutagenesis efficiency using serial injection and mismatch repair deficiency mice. PLoS One 11: e0159377.

Garcia‐Garcia MJ and Anderson KV (2003) Essential role of glycosaminoglycans in Fgf signaling during mouse gastrulation. Cell 114: 727–737.

Geister KA, Timms AE and Beier DR (2018) Optimizing genomic methods for mapping and identification of candidate variants in ENU mutagenesis screens using inbred mice. G3 (Bethesda, MD.) 8: 401–409.

Godinho SI, Maywood ES, Shaw L, et al. (2007) The after‐hours mutant reveals a role for Fbxl3 in determining mammalian circadian period. Science 316: 897–900.

Goldsworthy M, Bai Y, Li CM, et al. (2016) Haploinsufficiency of the insulin receptor in the presence of a splice‐site mutation in Ppp2r2a results in a novel digenic mouse model of type 2 diabetes. Diabetes 65: 1434–1446.

Hafezparast M, Klocke R, Ruhrberg C, et al. (2003) Mutations in dynein link motor neuron degeneration to defects in retrograde transport. Science 300: 808–812.

Hai T, Cao C, Shang H, et al. (2017) Pilot study of large‐scale production of mutant pigs by ENU mutagenesis. eLife 6: e26248.

Hardisty‐Hughes RE, Tateossian H, Morse SA, et al. (2006) A mutation in the F‐box gene, Fbxo11, causes otitis media in the Jeff mouse. Human Molecular Genetics 15: 3273–3279.

Jeans AF, Oliver PL, Johnson R, et al. (2007) A dominant mutation in Snap25 causes impaired vesicle trafficking, sensorimotor gating, and ataxia in the blind‐drunk mouse. Proceedings of the National Academy of Sciences of the United States of America 104: 2431–2436.

Justice MJ, Carpenter DA, Favor J, et al. (2000) Effects of ENU dosage on mouse strains. Mammalian Genome 11: 484–488.

Keays DA, Clark TG and Flint J (2006) Estimating the number of coding mutations in genotypic‐ and phenotypic‐driven N‐ethyl‐N‐nitrosourea (ENU) screens. Mammalian Genome 17: 230–238.

Knapik EW (2000) ENU mutagenesis in zebrafish – from genes to complex diseases. Mammalian Genome 11: 511–519.

Lewis MA, Quint E, Glazier AM, et al. (2009) An ENU‐induced mutation of miR‐96 associated with progressive hearing loss in mice. Nature Genetics 41: 614–618.

Lisse TS, Thiele F, Fuchs H, et al. (2008) ER stress‐mediated apoptosis in a new mouse model of osteogenesis imperfect. PLoS Genetics 4: e7.

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

Nadon NL, Strong R, Miller RA and Harrison DE (2017) NIA Interventions Testing Program: investigating putative aging intervention agents in a genetically heterogeneous mouse model. eBioMedicine 21: 3–4.

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.

Potter PK, Bowl MR, Jeyarajan P, et al. (2016) Novel gene function revealed by mouse mutagenesis screens for models of age‐related disease. Nature Communications 7: 12444.

Quwailid MM, Hugill A, Dear N, et al. (2004) A gene‐driven ENU‐based approach to generating an allelic series in any gene. Mammalian Genome 15: 585–591.

Rajaraman S, Davis WS, Mahakali‐Zama A, et al. (2002) An allelic series of mutations in the kit ligand gene of mice. I. Identification of point mutations in seven ethylnitrosourea‐induced Kitl(Steel) alleles. Genetics 162: 331–340.

Russell WL, Kelly EM, Hunsicker PR, et al. (1979) Specific‐locus test shows ethylnitrosourea to be the most potent mutagen in the mouse. Proceedings of the National Academy of Sciences of the United States of America 11: 5818–5819.

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

Van Boxtel R, Gould MN, Cuppen E and Smits BM (2010) ENU mutagenesis to generate genetically modified rat models. Methods in Molecular Biology 597: 151–167.

Vinuesa CG, Cook MC, Angelucci C, et al. (2005) A RING‐type ubiquitin ligase family member required to repress follicular helper T cells and autoimmunity. Nature 435: 452–458.

Zhang Z, Alpert D, Francis R, et al. (2009) Massively parallel sequencing identifies the gene Megf8 with ENU‐induced mutation causing heterotaxy. Proceedings of the National Academy of Sciences of the United States of America 106: 3219–3224.

Further Reading

Acevedo‐Arozena A, Wells S, Potter P, et al. (2008) ENU mutagenesis, a way forward to understand gene function. Annual Review of Genomics and Human Genetics 9: 49–59.

Crawley J (2007) What's Wrong With My Mouse?: Behavioral Phenotyping of Transgenic and Knockout Mice, 2nd edn. Hoboken, NJ: John Wiley & Sons, Inc.

Gondo Y (2008) Trends in large‐scale mouse mutagenesis: from genetics to functional genomics. Nature Review. Genetics 9: 803–810.

Silver LM (1995) Mouse Genetics. New York: Oxford University Press.

Simon MM, Moresco EM, Bull KR, et al. (2015) Current strategies for mutation detection in phenotype‐driven screens utilising next generation sequencing. Mammalian Genome 9–10: 486–500.

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Potter, Paul K(Jun 2018) Deconstructing Gene Function through ENU Mutagenesis. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0022409.pub2]