Elsevier

Molecular Brain Research

Volume 132, Issue 2, 20 December 2004, Pages 105-115
Molecular Brain Research

Research report
Large-scale mutagenesis of the mouse to understand the genetic bases of nervous system structure and function

https://doi.org/10.1016/j.molbrainres.2004.09.016Get rights and content

Abstract

N-ethyl-N-nitrosourea (ENU) mutagenesis is presented as a powerful approach to developing models for human disease. The efforts of three NIH Mutagenesis Centers established for the detection of neuroscience-related phenotypes are described. Each center has developed an extensive panel of phenotype screens that assess nervous system structure and function. In particular, these screens focus on complex behavioral traits from drug and alcohol responses to circadian rhythms to epilepsy. Each of these centers has developed a bioinformatics infrastructure to track the extensive number of transactions that are inherent in these large-scale projects. Over 100 new mouse mutant lines have been defined through the efforts of these three mutagenesis centers and are presented to the research community via the centralized Web presence of the Neuromice.org consortium (http://www.neuromice.org). This community resource provides visitors with the ability to search for specific mutant phenotypes, to view the genetic and phenotypic details of mutant mouse lines, and to order these mice for use in their own research program.

Introduction

The publication of the human genome sequence was a cause for celebration and sober assessment. While sequence was presented for the whole genome [17], [18], it was also clear that over 40% of the genes did not readily fit into a gene ontology category, and the vast majority of genes had little if any functional annotation. With the goal to understand the function of all the genes in the human genome, and the acknowledgment that the mouse was the best model organism to attain this objective, a variety of complementary strategies in the mouse has emerged as the means to this end [1], [10]. To understand the function of genes in the central nervous system is arguably one of the most challenging of these initiatives, as we are still learning how human neurological and behavioral phenotypes manifest themselves in mice. Until recently, neurological phenotypes in mice were detected in a cottage-industry setting at a steady but slow pace (e.g., investigators studying select phenotypes of mice with natural variants or spontaneous mutations or gene knockouts). The wonderful productivity of mutagenesis programs in fly, worm, and fish, and the demonstration by the German (http://www.gsf.de/ieg/groups/enu-mouse.html) and UK (http://www.mgu.har.mrc.ac.uk/mutabase/) groups that these efforts can be scaled to the mouse led NIH to support three mutagenesis centers in the United States to screen for mutations that affected various aspects of nervous system structure and function. The phenotype is the driving force in these efforts at Northwestern University, the Jackson Laboratory, and the Tennessee Mouse Genome Consortium (TMGC). These Centers are all using a forward-genetic strategy of combining chemical mutagenesis with high-throughput phenotypic screening, allowing the altered phenotypes of mutants to lead to their identification. Identification of mutants, in turn, allows for the association of genes with functions, as well as providing model systems for studying the molecular and genetic mechanisms underlying the affected phenotype.

Section snippets

ENU mutagenesis approach

The screens being carried out by the three mutagenesis centers employ the chemical supermutagen N-ethyl-N-nitrosourea (ENU). ENU is an ethylating agent that is both mutagenic and cytotoxic in mouse spermatogonial stem cells [16]. The average per-locus mutation frequencies achievable by ENU treatment, historically measured at visibly marked loci, depend on the dose, gene (target) size, and ability to recognize phenotype, and range from 1/1500 for a single 250 mg/kg dose to ∼1/700 for a

Genome-wide and regional approaches to mutagenesis

Two general approaches can be used with ENU mutagenesis. The more widely used approach is to target the whole genome (Fig. 1). The projects at Northwestern and the Jackson Laboratory are using ENU to produce mutations throughout the genome, followed by three generations of breeding to produce homozygous mutants. By screening third-generation (G3) progeny of mutagen-treated mice, dominant, semidominant, and recessive mutations may all be recovered. In this approach, the target is broad but the

Bioinformatic tools

The amount of information that is needed to be assembled and processed is quite substantial in such an animal- and screen-intensive program. To make each of the mutagenesis programs run successively, it has been critical to establish Web-based bioinformatic tools to manage the multitude of transactions that start from the time of mutagenesis, subsequent breeding of several generations, phenotype screens, identification of outliers, and then pushing the whole process through verification and

Neuromice.org—a community resource for neurological mutant mice

New mutant mouse lines are important tools for functional gene identification and only have their full scientific potential realized through investigation by the greater scientific community. In order to create an efficient and effective means to distribute mutant mice, a distribution consortium, known as Neuromice.org, was created.

The Neuromice.org consortium was established as part of the Trans-NIH Initiative to create new research resources, such as mutant mouse models of neurological and

Conclusions

The extension of ENU mutagenesis to specifically identify recessive mutant mice with neurological phenotypes has been a challenge in terms of building up the personnel and phenotyping expertise and the informatics infrastructure to help run the various programs. Each of the three facilities has developed unique ways to meet this challenge and have identified mutant mice with a wide range of neurobehavioral phenotypes. Furthermore, these efforts speak to an industrial-sized approach to

Acknowledgments

We would like to thank all the members of each Mutagenesis Center for their contributions to this community effort. This work is generously supported by cooperative agreements U01-MH61971 (TMGC), U01-MH61915 (NU), U01-NS41215 (TJL).

References (18)

  • M.F. Lyon et al.

    Use of an inversion to test for induced X-linked lethals in mice

    Mutat. Res.

    (1982)
  • J. Battey et al.

    An action plan for mouse genomics

    Nat. Genet.

    (1999)
  • C. Bult et al.

    A genome end-game: understanding gene function in the nervous system

    Nat. Neurosci.

    (2004)
  • K. Hentges et al.

    The flat-top gene is required for the expansion and regionalization of the telencephalic primordium

    Development

    (1999)
  • S. Hitotsumachi et al.

    Dose-repetition increases the mutagenic effectiveness of N-ethyl-N-nitrosourea in mouse spermatogonia

    Proc. Natl. Acad. Sci. U. S. A.

    (1985)
  • P. Isnard, N. Core, P. Naquet and M. Djabali, Altered lymphoid development in mice deficient for the mAF4...
  • A.M. Isaacs et al.

    A mutation in Af4 is predicted to cause cerebellar ataxia and cataracts in the robotic mouse

    J. Neurosci.

    (2003)
  • M.J. Justice et al.

    Mouse ENU mutagenesis

    Hum. Mol. Genet.

    (1999)
  • A. Kasarskis et al.

    A phenotype-based screen for embryonic lethal mutations in the mouse

    Proc. Natl. Acad. Sci. U. S. A.

    (1998)
There are more references available in the full text version of this article.

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