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, including the ability to delete genes or introduce more subtle single mutations. 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 throughout the genome in many model organisms. ENU mutagenesis is a powerful hypothesis‐generating approach currently being exploited to create new mouse models through both forward and reverse genetics approaches.

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.

  • ENU phenotype‐driven screens identify new mouse models by means of phenotyping.

Keywords: ENU; ENU mutagenesis; mouse; mouse models; phenotype; genetics

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 individual 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‐driven 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 to the identification of the causative mutation. Genedriven 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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Further Reading

Acevedo‐Arozena A, Wells S, Potter P et al. (2008) ENU mutagenesis, a way forward to understand gene function. Annual Review in 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: Wiley.

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

Web links

Ensembl: http://www.ensembl.org/index.html

MGI: http://www.informatics.jax.org/

Mouse SNP Jackson Laboratories database: http://phenome.jax.org/pub‐cgi/phenome/mpdcgi?rtn=snps/door

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Acevedo‐Arozena, Abraham, Ricketts, Thomas, and Corrochano, Silvia(Apr 2010) Deconstructing Gene Function through ENU Mutagenesis. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0022409]