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  • Review Article
  • Published:

Gene-trap mutagenesis: past, present and beyond

Nature Reviews Genetics volume 2, pages 756–768 (2001)Cite this article

Abstract

Although at least 35,000 human genes have been sequenced and mapped, adequate expression or functional information is available for only ∼15% of them. Gene-trap mutagenesis is a technique that randomly generates loss-of-function mutations and reports the expression of many mouse genes. At present, several large-scale, gene-trap screens are being carried out with various new vectors, which aim to generate a public resource of mutagenized embryonic stem (ES) cells. This resource now includes more than 8,000 mutagenized ES-cell lines, which are freely available, making it an appropriate time to evaluate the recent advances in this area of genomic technology and the technical hurdles it has yet to overcome.

Key Points

  • The thorough functional annotation of the mouse genome will require the use of a combination of mutagenesis strategies, including gene targeting, conditional mutagenesis, chromosomal engineering, high-throughput phenotypic screens of chemically mutagenized mice, and gene and promoter trapping.

  • Gene trapping is an insertional mutagenesis strategy used in embryonic stem (ES) cells that combines high-throughput random mutagenesis with molecularly defined mutations.

  • Gene-trap vectors contain a promoterless reporter gene. If the trap vector randomly integrates into an endogenous gene locus, a fusion transcript is generated from the upstream sequence of the endogenous trapped gene and the reporter. This fusion transcript can simultaneously report the expression of the trapped gene, mutate the gene and provide a sequence tag for cloning the mutated gene.

  • Gene trapping is a genetic tool that can be used to isolate mutations according to their resulting phenotype in vivo, to the sequence of the trapped locus and to its expression. However, some gene-trap strategies are limited to identifying only those trapped genes that are expressed in undifferentiated ES cells. To overcome this, and other limitations, new trapping vectors and strategies are being developed.

  • New trap vectors are continually being developed to increase trapping efficiency, to enhance the recovery of trapped sequence, to trap specific classes of protein, and/or to allow post-insertional modification of the trapped loci by combining trapping with conditional mutagenesis.

  • There are several international, large-scale gene-trap efforts now underway that will collectively generate tens of thousands of mutant ES cells that are, and will be, freely available to the scientific community.

  • Investigation of the mouse lines derived from these ES-cell libraries could eventually help scientists to dissect the function of thousands of mammalian genes.

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Figure 1: The basic trap vectors.
Figure 2: A comparison of β-gal, β-geo and polyA gene-trap vectors.
Figure 3: The secretory-trap vector.
Figure 4: The plasmid rescue vector.

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Acknowledgements

We thank the following investigators who have shared unpublished data and preprints to help make this review as current as possible: G. Hicks, E. Ruley, P. Soriano, W. C. Skarnes, J. Wrana, W. Wurst and K.-I. Yamamura. We thank the Canadian Institutes of Health Research and the Leukemia & Lymphoma Society for generous funding of gene-trap research to W.L.S. We apologize to the many colleagues who have provided valuable insights and large amounts of data in the gene-trap field whose manuscripts were not cited owing to strict space limitations.

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Authors and Affiliations

  1. Programme in Development and Fetal Health, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Room 983, Toronto, M5G 1X5, Ontario, Canada

    William L. Stanford, Jason B. Cohn & Sabine P. Cordes

  2. Department of Molecular and Medical Genetics, University of Toronto, Toronto, M5S 1A8, Ontario, Canada

    Jason B. Cohn & Sabine P. Cordes

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  1. William L. Stanford
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  2. Jason B. Cohn
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  3. Sabine P. Cordes
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Correspondence to William L. Stanford.

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Alexandra Joyner's lab

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William Stanford's lab

Glossary

INTRACISTERNAL A PARTICLE

(IAP). Endogenous, non-infectious retroviral element that can undergo transposition and act as a mutagen.

ALLELIC SERIES

A collection of discrete mutations that affect the same gene.

SATURATION MUTAGENESIS

This occurs when a mutagenesis screen recovers at least one mutation in every gene.

INSERTIONAL MUTAGENESIS

A strategy that uses the insertion of DNA to mutagenize genes at the insertion site. The inserted sequence acts as a tag from which to clone the mutated gene.

PRONUCLEAR INJECTION

DNA injected into a one-cell mouse embryo before a single nucleus forms, when a male pronucleus and a female pronucleus are still present. DNA is usually injected into the more-visible male pronucleus to generate a transgenic embryo.

NEOMYCIN RESISTANCE GENE

The bacterial neomycin phosphotransferase gene provides resistance to the amino-glycoside analogue, G418.

INVERSE PCR

This technique allows the DNA that flanks a region of known sequence to be amplified. Cleavage products from restriction digests are circularized and then amplified by PCR using primers to the known sequence.

LONG TERMINAL REPEAT

(LTR). A sequence repeated at both ends of a retroviral DNA that is required for retroviral insertion into its target genomic DNA.

β-GALACTOSIDASE

This enzyme, encoded by lacZ, is a commonly used reporter, as it can act on various substrates to produce colour or fluorescent reactions to indicate the expression pattern of a gene.

β-GEO

A fusion protein encoded by the lacZ gene, which encodes the marker β-galactosidase, and the neo gene, which confers neomycin resistance.

SPLICE ACCEPTOR SITE

Sequences from exon–intron boundaries that mediate mRNA splicing.

5′RACE

(5′ rapid amplification of cDNA ends). RACE is a PCR-based method for amplifying unknown cDNA sequences by using primers that correspond to known sequence.

DIPHTHERIA TOXIN

A toxin secreted by the bacteria Corynebacterium diphtheriae. A cell lineage can be ablated using diphtheria toxin, by driving its cDNA from a lineage-specific promoter.

SV40 T ANTIGEN

A viral oncogene from the polyoma virus that can bind the tumour suppressors p53 and retinoblastoma, which blocks their functions and leads to the immortalization of the cell.

GENOTYPIC SCREEN

A genetic screen of mutants based solely on the sequence of the mutated gene.

EXPRESSION SCREEN

A genetic screen of mutants based solely on the expression pattern of the mutated gene.

ROSA

(Reverse orientation splice acceptor). In the ROSA series of gene-trap vectors, the long terminal repeat (LTR) is in the reverse orientation to the trapping vector to ensure that the LTR does not interfere with the endogenous expression pattern of the trapped gene.

AGGREGATION CHIMAERAS

A simple and inexpensive technique to generate mouse strains derived from embryonic stem (ES) cells, in which clumps of about eight ES cells are fused with eight-cell-stage embryos, developing into morphologically normal embryos that contain cells derived from both the ES cells and the donor embryo.

EXPRESSION-TRAPPING SCREEN

A gene-trap screen that identifies clones with trapped events in genes expressed in specific lineages.

BICISTRONIC

A bicistronic message allows two different proteins to be translated from the same mRNA strand, usually from a promoter and an internal ribosomal entry site.

INDUCTION TRAPPING

A gene-trap screen that identifies clones with trapped events in specific signalling pathways.

LIPOPOLYSACCHARIDE

Component of the outer membrane of Gram-negative bacteria. LPS is commonly used to elicit B-cell proliferation and differentiation.

ENHANCED GFP

(eGFP). An autofluorescent 27-kDa protein, originally identified in the jellyfish Aequorea victoria, that has been mutated to enhance fluorescence in mammalian cells.

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Stanford, W., Cohn, J. & Cordes, S. Gene-trap mutagenesis: past, present and beyond. Nat Rev Genet 2, 756–768 (2001). https://doi.org/10.1038/35093548

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  • DOI: https://doi.org/10.1038/35093548

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