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A forward-genetic approach for analysis of the immune system

Nature Reviews Immunology volume 3, pages 463–471 (2003)Cite this article

Key Points

  • The identification of all mammalian genes through genome sequencing creates the possibility of understanding immune development and function in the context of the whole genome.

  • Forward-genetic strategies allow the assigning of function to gene products without previous knowledge of gene activity or expression. Importantly, many types of mutation might be uncovered, including, but not only, null alleles.

  • Chemical mutagenesis using n-ethylnitrosourea (ENU) is a robust and proven method for generating mutant animals with various immunological and other traits of interest.

  • Technological changes have made positional cloning a practical and rapid method for gene identification, including single base-pair mutations.

  • Numerous developmental and functional mutations have been identified from ENU-based screening programmes, in some cases with unexpected associations of gene and function.

  • Further exploitation of forward-genetic technologies will depend largely on the ability to screen for phenotype and function in the context of the whole animal.

Abstract

The completion of the genome sequences of both humans and mice challenges biologists to determine gene function on a vast, whole-organism scale. Both phenotype-based ('forward') and gene-based ('reverse') strategies are being developed to approach this issue. Forward-genetic approaches, however, provide the unique ability of assigning function to genes in an unbiased, global manner that is independent of previous assumptions about gene function. In this article, we compare various genetic technologies for their potential role in dissecting immune-system development and function, with particular emphasis on the worldwide efforts that use chemical mutagenesis as a forward-genetic strategy.

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Figure 1: A representative breeding scheme for ENU mutagenesis.
Figure 2: Mapping of ENU-induced mutations.
Figure 3: Genetic identification of ENU-induced mutations.
Figure 4: Screening assay design for ENU-mutagenesis programmes.

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Acknowledgements

We would like to thank M. Brunkow, G. Carlson and the Celltech ENU team.

Author information

Authors and Affiliations

  1. Discovery Biology, Celltech R & D Inc., 1631 220th St SE, Bothell, 98021, Washington, USA

    Mark W. Appleby & Fred Ramsdell

Authors
  1. Mark W. Appleby
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  2. Fred Ramsdell
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Corresponding author

Correspondence to Fred Ramsdell.

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DATABASES

LocusLink

CD4

CD83

LAT

Rag2

FURTHER INFORMATION

Mouse genome informatics website

Glossary

CRE–LOX TECHNOLOGY

A site-specific recombination system that is used to delete a gene in mouse cells using Cre recombinase. Two short DNA sequences (LoxP sites) are engineered to flank the target DNA. Expression of Cre recombinase leads to excision of the intervening sequence. Depending on the type of promoter, Cre can be expressed at specific times during development or by specific sets of cells, including embryonic stem cells.

SINGLE TANDEM REPEATS AND SINGLE NUCLEOTIDE POLYMORPHISMS

(STRs and SNPs). A short series of DNA sequences (STRs) or single base-pair changes (SNPs) that are inherited in a Mendelian manner. Approximately three to six million SNPs are thought to exist in the mammalian genome and a variety of techniques are available for their specific detection.

PHENODEVIANT

An animal that has a property or response outside the normal distribution for the other animals of the same pedigree. Phenodeviant animals are often presumed to result from the mutation of a specific gene, but this must be confirmed by gene identification.

EXPRESSED SEQUENCE TAG DATABASE

(EST database). A database (public or private) that catalogues genes that are presumed to be expressed at the RNA level in a particular cell type or tissue.

NON-OBESE DIABETIC MICE

(NOD mice). An inbred strain of mice that spontaneously develops T-cell-mediated autoimmune diabetes.

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Appleby, M., Ramsdell, F. A forward-genetic approach for analysis of the immune system. Nat Rev Immunol 3, 463–471 (2003). https://doi.org/10.1038/nri1109

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