Trends in Neurosciences
Volume 25, Issue 7, 1 July 2002, Pages 336-340
Journal home page for Trends in Neurosciences

Research update
Knockout mice: simple solutions to the problems of genetic background and flanking genes

https://doi.org/10.1016/S0166-2236(02)02192-6Get rights and content

Abstract

Inducing null mutations by means of homologous recombination provides a powerful technique to investigate gene function and has found wide application in many different fields. However, it was realized some time ago that the specific way in which such knockout mutants are generated can be confounding, making it impossible to separate the effects of the induced null mutation from those of alleles originating from the embryonic stem cell donor. In addition, effects from null mutations can be altered on different genetic backgrounds. Here we present some simple breeding strategies to test for flanking gene effects that are compatible with the recommendations of the Banbury Conference on Genetic Background in Mice and with common practices of creating and maintaining mouse knockout lines.

Section snippets

Interactions with genetic background and environment

Another possible complication in gene-knockout experiments is the fact that the phenotype resulting from a null mutation can depend on the general genetic background of mouse strains used for this research. Thus, congenic strains carrying the same null mutation can sometimes show widely divergent phenotypes, depending on the genotype of the recipient strain 7., 8.. Recent examples are the findings of Mineur et al. [9] and Ivanco and Greenough [10]. They described opposite effects of a null

The flanking gene problem: inadequacies of the Banbury Conference recommendations

The Banbury Conference discussed breeding strategies for maintaining targeted mutations in mouse strains, and how to deal with the genetic background problem when breeding mouse samples for testing [3]. Thus, it was recommended that targeted mutations be maintained by backcrossing the initial F1 carriers with mice of two different inbred mouse strains, eventually producing congenic and/or co-isogenic lines (Fig. 1, Box 1). This provides widely comparable mice that can be used for testing and,

Available strategies to solve the flanking-gene problem

The flanking-gene problem might be solved by abandoning the classic strategy used to generate targeted mutations, as there are alternative models that are unaffected. For example, in so-called ‘conditional knockout’ models, wild-type controls can be substituted with mutants that have the mutation in the ‘off’-state [16]. Alternatively, the normal phenotype of a constitutional null mutation could be rescued by re-introduction of the wild-type allele as a transgene [17]. Finally, one might use ES

Breeding strategies

Here we present some simple breeding strategies to deal with the flanking gene problem. The idea is to confirm the mutant phenotype by comparing littermates in which the alleles at flanking genes always come from the ES-cell donor strain, be this in wild-type or mutant animals. We present two such strategies. The first is a post-hoc strategy, for the majority of classic constitutional knockout mice, where a recessive (haplosufficient) or incompletely dominant null mutation is being conserved by

Conclusions

The proposed breeding schemes require no specific technology, little resources and no more time than is already needed to comply with the recommendations of the Banbury Conference. Importantly, the ‘reverse F2’ strategy permits post-hoc testing for flanking gene effects, even when backcrossing is done only to the C57BL/6 strain (now a routine procedure in most laboratories). The only inconveniences are the replication of neurobehavioral testing some time after initial characterization, and the

Acknowledgements

This work was supported by the Swiss National Science Foundation, the European Community (BIO4CT980297/ BBW98.0125) and the NCCR grant ‘Neural Plasticity and Repair’. We thank Robert T. Gerlai (Indianapolis, IN, USA) for critically reading an earlier version of the manuscript.

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