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Insertional mutagenesis in mice: new perspectives and tools

Key Points

  • Recent initiatives have outlined the strategies and goals of the mouse community to achieve complete functional annotation of the mouse genome.

  • Insertional mutagenesis strategies have proved invaluable for mouse genetics research, and new technologies aim to improve the usefulness of this approach.

  • The Velocigene system, developed by Regeneron Pharmaceuticals, uses a BAC recombination system to generate targeted knockouts faster and more efficiently than traditional gene targeting systems.

  • The mutagenic and chromosome engineering resource (MICER) is a resource of insertion vectors that can target specified genes for mutation with great efficiency and can allow the production of templates for chromosome engineering.

  • The progress of genome-wide gene-trap screens has been substantial, and the advent of expression screening allows for prescreening in culture for gene classes of interest.

  • Using the Cre/loxP system or exploiting the increased incidence of non-sister chromatid exchange in Bloom syndrome cells allows the induction of mitotic recombination in embryonic stem cells. These approaches can be used to generate biallelic homozygous mutations in culture for recessive genetic screens.

  • The optimization of several transposable elements has led to the development of efficient systems for in vivo transposon mutagenesis for forward genetic screens. These elements can also potentially be used for generating substrates for chromosome engineering, gain-of-function screens and somatic cell insertional mutagenesis in the mouse.

  • A synthesis of insertional mutagenesis technologies will be necessary to achieve complete genome coverage for functional annotation, and will compliment alternative approaches including chemical mutagenesis.

Abstract

Insertional mutagenesis has been at the core of functional genomics in many species. In the mouse, improved vectors and methodologies allow easier genome-wide and phenotype-driven insertional mutagenesis screens. The ability to generate homozygous diploid mutations in mouse embryonic stem cells allows prescreening for specific null phenotypes prior to in vivo analysis. In addition, the discovery of active transposable elements in vertebrates, and their development as genetic tools, has led to in vivo forward insertional mutagenesis screens in the mouse. These new technologies will greatly contribute to the speed and ease with which we achieve complete functional annotation of the mouse genome.

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Figure 1: Insertional mutagenesis approaches and mechanisms.
Figure 2: Expression trapping in embryonic stem cells.
Figure 3: Induced mitotic recombination using Cre/loxP or a Bloom syndrome mutant background.
Figure 4: Exploiting the 'local hopping' of the Sleeping Beauty transposon.

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Authors and Affiliations

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Corresponding author

Correspondence to David A. Largaespada.

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Competing interests

David Largaespada is a co-founder and part owner of the biotechnology company Discovery Genetics Inc (DGI). DGI has licensed Sleeping Beauty technology from the University of Minnesota. DGI is pursuing the use of Sleeping Beauty for human gene therapy.

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FURTHER INFORMATION

Ensembl

International Gene-trap Consortium

MICER

NCBI

Omnibank

Regeneron Pharmaceuticals

The Arnold and Mabel Beckman Center for Transposon Research

Nature Biotechnology

Glossary

ES CELL LIBRARY

A collection of embryonic stem cell clones that contain a mutation at a distinct location of the genome.

INSERTIONAL MUTAGENESIS

A mutation caused by the addition of DNA to effectively disrupt or alter the function of a given gene.

ALLELIC SERIES

A panel of multiple mutations at distinct locations within the same gene, sometimes resulting in phenotypes that vary in severity or that are completely distinct.

CHROMOSOME ENGINEERING

The controlled generation of chromosomal deletions, inversions or translocations with defined endpoints.

NONSENSE-MEDIATED mRNA DECAY

The process by which the cell destroys mRNAs that are untranslatable owing to the presence of a nonsense codon within the coding region.

EXPRESSION TRAPPING

The identification of ES cell clones from a library that contains gene-trap integrations within genes that are transcriptionally responsive to specific exogenous stimuli.

HAPLOINSUFFICIENCY

The inability for the remaining wild-type allele to compensate for a heterozygous loss-of-function mutation.

PRODRUG METRONIDAZOLE

A compound that when activated by nitrosoreductase is converted into a toxic alkylating agent.

BLOOM SYNDROME

Human autosomal recessive disorder caused by a loss-of-function mutation in a RecQ helicase. It is characterized by genomic instability, increased incidence of sister chromatid exchange, and predisposition to an array of tumour types.

RETROTRANSPOSONS

Transposable elements that mobilize by a 'copy and paste' mechanism. Retrotransposons are transcribed and then reverse transcribed by element-encoded enzymes. The resulting elements are subsequently integrated in the genomes, at a new location (for example, LINE-1 and intracisternal A particle).

DNA TRANSPOSONS

Elements that mobilize by a 'cut and paste' mechanism with the transposon DNA being excised from its original location and inserted elsewhere by the transposase enzyme.

TC1/MARINER TRANSPOSONS

DNA-based 'cut and paste' transposable elements found in numerous host organisms and having a small target sequence for integration (for example, Sleeping Beauty, Himar, Minos).

CONTIGUOUS GENE SYNDROME

A multi-symptom disorder that is caused by a deletion of a large sequence of DNA that encodes several genes.

BALANCER CHROMOSOME

A chromosome with an inversion (or multiple inversions) of an interval of DNA that represses meiotic recombination.

HYPOMORPHIC ALLELE

A mutation in a gene that results in reduced expression or activity of the gene without complete loss of function.

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Carlson, C., Largaespada, D. Insertional mutagenesis in mice: new perspectives and tools. Nat Rev Genet 6, 568–580 (2005). https://doi.org/10.1038/nrg1638

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