REVIEWS GENE-TRAP MUTAGENESIS: PAST, PRESENT AND BEYOND William L. Stanford*, Jason B. Cohn*‡ and Sabine P. Cordes*‡ 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. MOUSE GENOMIC TECHNOLOGIES INTRACISTERNAL A PARTICLE (IAP). Endogenous, non- In Simone de Beauvoir’s existentialist novel All Men Are low frequency at which spontaneous mutations occur infectious retroviral element Mortal, two creatures attain immortality: a man who (~5 × 10−6 per locus) makes dissecting an entire genetic that can undergo transposition intends to be an enlightened ruler and a circling mouse. pathway by awaiting the appearance of spontaneous and act as a mutagen. Clearly, this was a mutant mouse — perhaps a descen- mutations unfeasible in a normal human lifespan or ALLELIC SERIES dant of the Chinese waltzing mice that were first with the resources of an averagely sized mouse house. In A collection of discrete described several thousand years ago. de Beauvoir’s addition, the molecular lesions that are responsible for mutations that affect the same would-be king chose the mouse as his companion for spontaneous mutations can range from small base gene. eternity for the same reason that we are pursuing mouse changes in coding sequences to retroviral insertions, to mutants today: the mouse provides us with an effective regulatory mutations of unknown character. For exam- model of ourselves, be it for testing potions of immortali- ple, several spontaneous mutant alleles of the agouti ty or for understanding human disease and development. coat colour locus (a), such as a16H and ae, are caused by Mouse fanciers have collected spontaneous mouse mutations in the coding region3, whereas others (Ahvy, mutants for millennia. Regrettably, the spontaneity of Aiy and Avy) are caused by the integration of a retrovirus- their appearance has often been matched, if not sur- like element — the INTRACISTERNAL A PARTICLE (IAP) — into passed, by their impromptu disappearance. As genetics different positions upstream of the transcriptional start *Programme in 4,5 Development and Fetal became a formalized science, the collection and analysis site, which deregulates agouti expression . So, although Health, Samuel Lunenfeld of spontaneous mutants became more systematic. the unpredictable molecular nature of spontaneous Research Institute, Today some large mouse colonies, such as those at The mutations is valuable for generating ALLELIC SERIES, these Mount Sinai Hospital, Jackson Laboratory, have regular, in-house meetings — spontaneous events do little to expedite gene identifica- 600 University Avenue, so-called mouse circuses — in which the newest sponta- tion, and they do not allow mouse models of human Room 983, Toronto, Ontario, Canada M5G 1X5. neous mutants make their debut. By nature of their disorders to be specifically designed. Many mouse ‡Department of Molecular identification, these mutants usually have strikingly visi- mutagenesis strategies have evolved to address these and Medical Genetics, ble phenotypes. Over time, numerous spontaneous shortcomings, and each generates mutations of a differ- University of Toronto, mutants, such as the white spotted (W) mutation, which ent molecular nature and at varying frequencies, as Toronto, Ontario, Canada M5S 1A8.Correspondence was first identified by its white belly spot and which shown in TABLE 1. Together, these strategies should allow to W.L.S. email: affects haematopoiesis, have lent credence to the mouse us to design ideal mouse models for studying human [email protected] as a clinically relevant model organism1,2.However,the disease and genetic pathways in vertebrates. In this 756 | OCTOBER 2001 | VOLUME 2 www.nature.com/reviews/genetics © 2001 Macmillan Magazines Ltd REVIEWS Table 1 | A comparison of mutagenesis strategies Mutagenesis Mutagenesis Type of mutation induced Primary advantages Primary disadvantages strategy (type) frequency Spontaneous 5 × 10−6 per locus Point mutations, small deletions, Visible phenotypes; only Only visible phenotypes detected, chromosomal rearrangements, requirement is observant at very low frequency. and insertions of endogenous mouse handlers. retrovirus-like sequences. X-ray 13–50 × 10−5 Chromosomal rearrangements: Rearrangements act as a Multiple genes affected, hard to per locus ranging from simple deletions, molecular landmark for cloning. dissect individual gene function. inversions and translocations, to complex rearrangements. Chlorambucil 127 × 10−5 Chromosomal rearrangements, Same as X-ray, but higher Multiple genes affected, hard to (chemical per locus especially smaller deletions mutagenesis frequency. dissect individual gene function. mutagen) (100–500 kb) and translocations. Ethylnitrosourea 150 × 10−5 Primarily generates point Single-gene mutations, amenable No molecular landmarks for (chemical per locus mutations, occasionally very to high throughput. cloning. mutagen) small deletions (20–50 bp). Transgene/ 5–10% of Disrupts endogenous gene Provides a molecular landmark Labour-intensive, not applicable retroviral transgenic animals expression or coding sequence. for cloning. to high-throughput approaches. (insertional Sometimes causes chromosomal mutagen) rearrangements. Gene targeting Almost 100% of Generates insertions or deletions, Can design type of mutation as Requires knowledge of gene and (insertional transgenic animals* as designed. required. its structure, labour-intensive, mutagen) unpredictable phenotypes. Trapping Almost 100% of Disrupts endogenous Forward-genetic strategy, easy Unpredictable phenotypes. (insertional transgenic coding sequence. to clone mutated gene, reports mutagen) animals* endogenous gene-expression pattern. * Requires pre-screening of embryonic stem cells in vitro. review, we discuss the development of gene-trap muta- Chemical mutagenesis with ethylnitrosourea (ENU), genesis and place it in context with other mouse func- which primarily introduces point mutations into sper- tional genomic techniques. We also summarize the matogonial stem cells8, is a preferred approach to the field’s progress and impediments, and point to some above strategies as ENU-induced mutations usually potential directions that gene trapping might take after affect only single genes. However, ENU mutagenesis SATURATION MUTAGENESIS has been achieved. provides no molecular landmarks with which to recover mutated genes. Because, in addition to the advantages Mutagenesis strategies listed in TABLE 1, ENU is easy to administer, and because On considering the pitfalls of spontaneous mutations, ENU-treated males can be used to generate mutant mouse geneticists sought to identify high-efficiency progeny for many months, this mutagenesis strategy has mutagenesis strategies that could produce mutations of gained considerable popularity9. a relatively defined molecular nature. Towards this end, In 1976, the introduction of exogenous retroviral 10 SATURATION MUTAGENESIS the first X-ray mutagenesis experiments on the mouse DNA into the mouse germ line was first reported , and This occurs when a mutagenesis were carried out in the 1930s, and gained momentum as INSERTIONAL MUTAGENESIS began to be hotly pursued in the screen recovers at least one larger centres (such as Oakridge, Tennessee,USA, and mouse. But because retroviral infection usually occurred mutation in every gene. Harwell, Oxford, UK) began to study the effects of radi- after the one- or two-cell stage of embryonic develop- 6 INSERTIONAL MUTAGENESIS ation fall-out on genetic stability . The frequency of X- ment, multiplicity of infection was extremely variable A strategy that uses the ray-induced mutations is 20–100 times greater than that and could lead to the generation of chimeric animals. insertion of DNA to of spontaneously occurring mutations. Furthermore, X- However, a mutation could be recovered by additional mutagenize genes at the ray mutagenesis causes chromosomal rearrangements, breeding if an insertion was transmitted through the insertion site. The inserted which can provide a molecular landmark for identifying germ line11,12. Despite the problems associated with this sequence acts as a tag from which to clone the mutated the affected gene(s). However, several genes are often approach, the insight that retroviral insertion could alter gene. affected by these chromosomal rearrangements and, as endogenous genes and their expression, leading to a result, their type and complexity is difficult to control. tumorigenesis and leukaemia10 in mutagenized mice, PRONUCLEAR INJECTION Similar multigene deletions and chromosomal translo- spurred the cloning of retroviral insertion sites to recover DNA injected into a one-cell mouse embryo before a single cations can be generated at a greater frequency by muta- the affected, tumorigenic genes, and validated the poten- 7 nucleus forms, when a male genizing mice with the chemical chlorambucil . tial usefulness