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NATURE REVIEWS | GENETICS VOLUME 1 | NOVEMBER 2000 | 109

identify the interrupted gene have been used to speedup the gene disruption process3.

Mutation isolation by the mouse supermutagen N-ethyl-N-nitrosourea (ENU) has the potential to generatean abundance of mutations in phenotype-driven screens(BOX 1). Mutations induced by ENU provide a uniquemutant resource. Although loss-of-function mutationsare valuable, an important advantage of chemical muta-genesis is that it generates ALLELIC SERIES. These provide arange of phenotypes in which a gene’s function can beaffected in different ways. Loss-of-function mutations,viable HYPOMORPHS of lethal COMPLEMENTATION groups, ANTI-

MORPHS, and gain-of-function mutations have been iso-lated in mouse mutagenesis screens. MISSENSE MUTATIONS incoding regions are common after ENU treatment, givingmany different phenotypes and providing a fine-struc-ture dissection of protein function and complex mam-malian isoforms. Furthermore, phenotype-driven muta-genesis offers many advantages in speed of generationand removal of investigator bias over allelic series gener-ated by homologous recombination.

Dominant mutations: large-scale isolationTwo recent papers4,5 published in Nature Genetics illus-trate the power of phenotype-driven mutagenesisapproaches in the mouse. One screen, directed by SteveBrown at Harwell, UK, is being carried out by a consor-tium of the Medical Research Council and Smith KlineBeecham Pharmaceuticals (UK ENU Mutagenesis pro-gramme). The second, led by Rudi Balling and MartinHrabe de Angelis at the GSF Forschungszentrum für

The completion of the human genome sequencepromises a new era for biology. Although manyapproaches to functional studies will provide new infor-mation, the approach that has defined function in manyorganisms is mutagenesis. Recently, the mouse hasjoined other model organisms, such as C. elegans,Drosophila and zebrafish, in large-scale mutation isola-tion. This review summarizes the current mouse muta-genesis efforts and compares them with programmes inother organisms. It also discusses the mutagenesisefforts that are underway and the need for infrastruc-ture to support the analysis, maintenance, archiving anddistribution of the new mutants.

Mouse mutagenesisThe biological similarity and extensive comparativegenetic linkage map between mouse and human makesthe mouse an ideal model organism for defining mam-malian gene function and modelling human disease. Alarge array of genetic tools with which to investigate themouse genome is available, and all are being used.These genetic approaches can be genotype-driven, asgene disruptions are, or phenotype-driven, as tradi-tional genetic screens are. For example, by usingembryonic stem (ES) cell technology, any gene involvedin human disease can be disrupted, providing valuableinformation about the consequences of mutation inthe whole animal1; and to investigate gene functionsthrough expression patterns, enhancer-driven GENE-TRAP

approaches have been devised2. More recently, gene-trap vectors that provide a valuable sequence tag to

CAPITALIZING ON LARGE-SCALEMOUSE MUTAGENESIS SCREENSMonica J. Justice

Variation is the crux of genetics. Mutagenesis screens in organisms from bacteria to fish have provided a battery of mutants that define protein functions within complex pathways.Large-scale mutation isolation has been carried out in Caenorhabditis elegans, Drosophilamelanogaster and zebrafish, and has been recently reported in the mouse in two screens that have generated many new, clinically relevant mutations to reveal the power of phenotype-driven screens in a mammal.

Department of Molecularand Human Genetics,Baylor College of Medicine,1 Baylor Plaza, Houston,Texas 77030, USA. e-mail:[email protected]

GENE TRAPPING

A mutation strategy that usesinsertion vectors to trap orisolate transcripts from flankinggenes. The inserted sequenceacts as a tag from which to clonethe mutated gene.

ALLELIC SERIES

An array of possible mutantforms of a gene, which usuallycause multiple phenotypes.

HYPOMORPH

A mutant allele that does noteliminate the wild-typefunction of a gene and may givea less severe phenotype than aloss-of-function mutant.

COMPLEMENTATION

When two mutations arecombined in an organism andthe phenotype is wild type, themutations are said tocomplement each other.

© 2000 Macmillan Magazines Ltd

110 | NOVEMBER 2000 | VOLUME 1 www.nature.com/reviews/genetics

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Umwelt und Gesundheit is a consortium of investigatorsfrom around Munich, Germany (German ENU-mousemutagenesis screen project). Together, these groups haveisolated over 300 confirmed mouse mutations and, byestimation from the number of inherited phenotypes,probably around 1,000 to 1,500 new mutations. Bothgroups used a simple breeding scheme to isolate domi-nant mutations (FIG. 1), screening 26,047 and 14,000mice, respectively, for visible phenotypes. A commonlyisolated class of mutations in a screen for dominant phe-notypes is the HAPLOINSUFFICIENT CLASS, and these loci yieldnumerous alleles in such a screen. Mutants that producea visible phenotype by dominance or haploinsufficiencymay already be present in the mutant database. However,each group used a unique screen on a subset of these ani-mals to isolate new classes of mutant phenotypes. TheHarwell group focused on sensory, neurological andneuromuscular phenotypes by using an assay calledSHIRPA6, as well as behavioural assays such as locomo-tor activity (LMA) and pre-pulse inhibition of theacoustic startle response (PPI). The German groupfocused on blood assays for haematological, clinicalchemistry, immunological and allergy defects. Eachgroup therefore identified a number of phenotypes rele-vant to human disease (TABLE 1).

A large proportion of the new mutations cause visi-ble defects. These include mutations that affect themouse hair and skin, pigmentation, skeletal morpholo-gy and eyes. The most common phenotype falls into thepigmentation class. This is not surprising, as a mousecoat colour is an easy phenotype to score. However, it isnot clear how many of these mutations will be uniquebecause mouse coat-colour variation has been the pur-suit of hobbyists and experimentalists for over 200years. Notably, the Harwell group complementationtested and mapped some of the mutations that seemedto be repeated phenotypes, and showed that two muta-tions were alleles of the Kit oncogene (Kit), and fourwere alleles of the Kit ligand (Kitl). The first allele of Kit,dominant white spotting (KitW), was first reported in1937 (REF. 7) and repeat mutations in Kit have occurred

a Cross 1:

b

Xa b c d

a b C d

a b c d

G1

ENU

*

a b C d*

a b c d

a b c d

Screen G1 offspring

VisibleSHIRPA

Harwell

Behaviour:LMA/PPI

VisibleBlood1. Haematology2. Immunology3. Allergy4. Clinical chemistry

GSF

Nociceptive

Figure 1 | Dominant screens to isolate new human disease models. a | Male mice weremutagenized with ENU and mated to wild-type females. Loci in a region of a chromosome aredesignated ‘a b c d’ (shown as black in the ENU mutagenized male and as green in the wild-type female). A new dominant mutation (C*, in red) is shown in the G1 offspring. b | G1 progenywere screened by both groups for visible and specialized phenotypes. Harwell included screensfor neurological disorders using the SHIRPA protocol. SHIRPA is a comprehensive stepwiseassessment of many parameters, designed to detect sensory, neuromuscular and neurologicaldefects6. The locomotor activity (LMA) test measures the number of times that a mouse splits anelectronic beam by rearing and the number of times that it crosses the cage over a 30-minuteperiod. The pre-pulse inhibition of the acoustic startle response (PPI) test takes place in a sound-proof chamber with calibrated noises delivered in random order over a time period. Startleresponses are measured after a single pulse alone, or after pre-pulses and a pulse. The GSFprogramme included screens for haematological, immunological and allergy defects, as well asfor nociception (pain response). Both groups included clinical chemistry screens; however, thesescreens were carried out on only a subset of animals.

Box 1 | ENU, a powerful mutagen in mice

N-ethyl-N-nitrosourea (ENU) is an alkylating agent that causes random single-base-pair mutations in a wide variety of organisms. The ethyl group of ENU can be transferred to oxygen or nitrogen molecules in DNA, causing an ethylated base to be mistakenly identified during DNA replication. Mispairing and base-pair substitution willresult if the mismatch is not repaired. In the mouse, the highest mutation rates occur in pre-meiotic spermatogonialstem cells, with single-locus mutation frequencies of 6 x 10-3 to 1.5 x 10-3 per mutagenized genome35,36, equivalent to isolating a new functionally defective allele in 175 to 655 screened gametes.

Mouse mutagenesis with ENU is simple. Male mice are injected intraperitoneally to mutagenize theirspermatogonial stem cells. Males go through a sterile period, but mutagenized spermatogonia eventuallyrepopulate the testis to produce clones of mutagenized sperm. Males are mated in breeding schemes to isolatemutations37. In dominant screens, many of the observed phenotypes (up to 50%) are not inherited, sophenotypes that result from mutation must be confirmed by breeding.

The primary lesions induced by ENU in the mouse germline are A•T to T•A transversions and A•T to G•Ctransitions. Of the sequenced mutations, 64% cause missense mutations, 10% cause NONSENSE MUTATIONS and 26%cause splicing errors38. ENU’s power lies in its efficiency in inducing functionally defective mutations. So it is themutagen of choice in phenotype-driven screens. However, disadvantages to its use include the difficulty oflocating and isolating the DNA lesion responsible for the desired phenotype, and its ability to make extraneousor undesired lesions elsewhere in the genome.

ANTIMORPH

A mutant allele that antagonizesgene function and acts in asemi-dominant manner.

MISSENSE MUTATION

A mutation that results in thesubstitution of an amino acid ina protein.

NONSENSE MUTATION

A mutation that results in theintroduction of a stop codon tocause the prematuretermination of the protein.

HAPLOINSUFFICIENT

A phenotype that arises indiploid organisms owing to the loss of one functional copy of a gene.



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must be localized so that candidate genes and relevanthuman disease models can be identified. Point muta-tions isolated by their phenotype must be mapped inmeiotic crosses using phenotypic information, as a mol-ecular tag is not available with this strategy. The Harwellconsortium tackled the mapping problem by incorpo-rating polymorphisms into their initial breeding strategy, and by using speed BACKCROSSES generated by invitro fertilization (IVF), along with DNA POOLING strate-gies. Speed backcrosses use sperm from an affected maleto fertilize eggs obtained from superovulated wild-typefemales, a quick and efficient way to obtain numerousprogeny for mapping dominant mutations. DNA pool-ing of mutant DNAs and wild-type DNAs eliminatesthe need to do molecular genome scans on individualbackcross offspring10,11. These methods improve the effi-ciency of mapping but do not eliminate the bottleneckof mutations that remain to be mapped. The develop-ment of mapping techniques that do not require exten-sive breeding, or mutation-isolation schemes that use amutation ‘tag’, would help to speed up the steps betweenidentifying a mutant phenotype and isolating theunderlying mutation.

The mouse already has a rich resource of visibledominant and recessive mutations from historical col-lections, spontaneous events, and smaller mutagenesisscreens. So it is unclear how many of these dominantENU-generated mutations will identify novel loci, andhow many represent alleles of known genes. After theloci have been mapped, identifying them should bemade simpler by the availability of the completesequence of the mouse and human genomes. Althoughsome hurdles remain, these mutagenesis efforts showthe efficacy of ENU in producing numerous new muta-tions in the mouse genome, especially if new phenotypescreens are incorporated. These single-gene mutationswill also be valuable for dissecting multigenic and mul-tifactorial traits12. Some of the mutations, for example,may represent a more penetrant allele of a locus thatcontributes to a multifactorial phenotype, but themonogenic trait will be easier to access molecularly.Placing the new mutations on different genetic back-grounds may also reveal different phenotypes, whichcould lead to the discovery of interacting loci in newdevelopmental, biochemical or physiological pathways.

Mutagenesis screens in other speciesLarge-scale mutation isolation has been carried out inworms, flies and zebrafish. Chemical mutagenesis in allorganisms is plagued by extraneous mutations, and so

frequently8. The first allele of Kitl was observed as asemi-dominant steeloid coat9, and mutations at thislocus have also appeared frequently. So their isolation inthis new mutant screen is not surprising.

The Harwell consortium confirmed more mutationsin the visible neurological class, whereas the Germangroup confirmed more mutations in the skeletal dys-morphology class, possibly reflecting the primary inter-ests of the two groups. Each group included a hearingtest and isolated a similar number of mutations affect-ing hearing per mutagenized genome. Six of theHarwell circling, deaf mutations were mapped to theproximal end of mouse chromosome 4, near the mousemutation Wheels (Whl), and all may be alleles of thesame locus. This implies that certain loci may be easilydetected in a screen for dominant phenotypes, and rais-es the possibility that this locus is haploinsufficient. Italso implies that this screen may already be approachingsaturation for some of the visible phenotypes.

Perhaps the most intriguing mutations are those thatwere isolated by the unique phenotype screens,designed to model human diseases. Harwell confirmeda total of 50 neurological mutations: 12 that were classi-fied as neurological and neuromuscular, 16 that causedcircling and deafness, and 22 that produced behaviouralphenotypes. The 22 behavioural mutations would nothave been isolated without the more thorough pheno-typic assessments by the SHIRPA, LMA and PPI tests.High or low LMA is associated with neurobehaviouralabnormalities, and deficits in PPI are seen in severalhuman psychiatric disorders, such as schizophrenia. TheGerman screen confirmed 54 haematological muta-tions: 15 that were found by complete blood counts, 30that caused immunological defects, and 9 that resultedin high or low serum immunoglobulin E (IgE) levels.The most frequent type of allergic reaction, immediatetype I hypersensitivity, is associated with elevated IgElevels. The immunological mutations were identified byscreens that utilized ELISA for immunoglobulins andantibodies against DNA, and by flow cytometry onperipheral blood to measure T- and B-cell populations.Clinical chemistry screens by both groups may have alsoisolated models of kidney disease. It is likely that manyof these mutations have identified novel gene functionsor novel genes.

Each group has a huge number of unconfirmedmutations and numerous unmapped mutations, sug-gesting that mutation confirmation and chromosomallocalization are bottlenecks in this mutation-isolationprocess. To be valuable, new ENU-induced mutations

Table 1 | Mutant phenotypes confirmed in recent mouse ENU screens

Phenotype classification

Skin/ Sensory Skeletal Size Neurological/ Haematological Immunological Allergy Clinical Nociceptivecoat organs behavioural chemistry

Harwell* 27 26 15 17 34 0 0 0 6 0

Germany‡ 31 18 40 4 21 15 30 9 8 9

Total 58 44 55 21 55 15 30 9 14 9

In addition, Harwell* confirmed one mutation that caused abdominal swelling owing to peritoneal ascites, and the German group‡ confirmed one mutation that affected the teeth.

ELISA (ENZYME-LINKED

IMMUNOSORBENT ASSAY.)

A sensitive antibody-basedmethod for the detection of anantigen such as a protein.

BACKCROSS

The mating of an individualwith its parent, or with anindividual of the same genotypeas its parent, to follow theinheritance of alleles andphenotypes.

DNA POOLING

A mapping strategy that poolsDNA from phenotypicallydistinct backcross or intercrossprogeny to identify markeralleles that are linked to thegenes that determinephenotype; it reduces the timeand expense of genotypingindividual mice from linkagecrosses.



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large-scale efforts have used transposable elementswhen possible. The worm genome has been elegantlytackled by both FORWARD and REVERSE GENETIC approachesthat have yielded millions of mutations13,14. Chemicalmutagenesis in fly and fish using ethyl methanesulphonate (EMS) or ENU, respectively, has been themethod of choice in many phenotype-driven screens toaddress gene function.

In 1984, the large-scale isolation of Drosophila muta-tions by chemical mutagenesis was reported by EricWieschaus and Christine Nusslein-Volhard15–17. Thesescreens capitalized on methodologies pioneered byMadeleine Gans18 and used a quick-screening assay tolook at Drosophila embryo morphology. Males withmarked chromosomes were treated with EMS and wereused in mating schemes designed to isolate recessivemutations, one chromosome at a time. The use of bal-ancer chromosomes to isolate recessive lethal mutationswas key to their success (BOX 2). Over 20,000 lethal muta-tions on the X, second and third chromosomes wereretrieved, and the embryonic-lethal group of mutantswas screened to reveal nearly 600 mutations that affect-ed the patterning of the larval cuticle. Using the powerof Drosophila genetics, each of the mutations was

mapped and tested for complementation. The utility ofthese mutations in dissecting the principles of embry-onic pattern formation has been unsurpassed19. Manyother mutagenesis screens have now been carried out inthe fly and, at present, the Drosophila stock centre hous-es over 8,000 stocks that include deletions, inversions,duplications, marked chromosomes, and insertionaland point mutations.

It has been argued that detailed studies in compara-tively simple model organisms, such as Drosophila, couldcondense the function of all human genes into a few hun-dred biological processes20. However, although studies inDrosophila will help to elucidate many genetic pathways,flies are limited as a model organism for analysing humangene function. There are two key reasons for this. First,health-related genes, such as those involved in arthritis,cardiovascular disease, asthma, inflammation, obesity,osteoporosis, fetal–maternal circulation and otheruniquely mammalian diseases, cannot be easily studied inlower organisms. Second, the network of interactionsmay be different between flies and mammals because ofthe many tissues that distinguish the two species.

In 1996, large-scale mutagenesis programs reportedrecessive mutations that affected developmentalprocesses in the zebrafish, Danio rerio. Together, twogroups identified a total of 6,647 mutations21,22. Theseprogrammes screened for embryogenesis defects andessential functions, phenotypes that are largely ignoredin the current mouse screens. Fish have the advantageof PARTHENOGENETIC activation of embryos, eliminatingthe requirement for complex breeding schemes to char-acterize some of the mutations. Furthermore, becausezebrafish eggs develop externally, they can be easilyvisualized, unlike the development of mice andhumans, which takes place in an uterine environment.Zebrafish embryos are also relatively transparent ,which aids the detection of phenotypic abnormalities.Mapping crosses in zebrafish are simple and cost effec-tive because many fish can be reared in a single tank.However, the zebrafish does not have an extensive com-parative molecular genetic linkage map with thehuman genome, although mapping efforts reveal thatlinkage groups are highly conserved between the twospecies23. Unfortunately, so many mutations were iso-lated in these initial screens that many of the newmutants without obvious morphological defects were

a Cross 1:X

a b c d

a b c d

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G1

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d Genotypesfrom cross:

*

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a b c d

a b c d

a b c d

a b c d

a b c d

a b c d

a b c d

a b c d a b c d

a b c d

Figure 2 | A three-generation breeding scheme without genetic markers to isolaterecessive mutations. Loci in a region of a chromosome are designated ‘a b c d’ (shown asblack in the ENU-mutagenized male and as green in the wild-type female). a | Fertile ENU-mutagenized males are mated to wild-type females. b | G1 males carrying a recessive mutation(c*, in red) are mated to wild-type females. c | G2 siblings are intercrossed, or G2 daughters,which may be wild type or carriers of a new recessive mutation, are mated back to theirfathers. d | New recessive mutations (c*/c*) can be identified in the G3 offspring.

FORWARD GENETICS

A genetic analysis that proceedsfrom phenotype to genotype bypositional cloning or candidate-gene analysis.

REVERSE GENETICS

A genetic analysis that proceedsfrom genotype to phenotype bygene-manipulation techniques,such as homologousrecombination in ES cells.

PARTHENOGENESIS

The production of offspring bya female with no geneticcontribution from a male.

Box 2 | Balancer chromosomes

• An ideal balancer chromosome suppressesrecombination over the length of a chromosome.Many balancers are constructed by making severalinversions on a single chromosome.

• The balancer chromosome should be easilyidentified in crosses by using a dominant marker.

• In self-sustaining crosses, balancer chromosomesshould be lethal when homozygous. Therefore, bymaintaining a lethal mutation in trans to thebalancer, the only offspring that will be viable froman intercross will be those that carry the mutationand are heterozygous for the balancer chromosome.



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spectrometry, can detect various metabolic disorders thataffect lipids, fatty acids or amino acids. Through grantsissued by the United States National Institutes of Health(NIH), more phenotypic assays are being developed toidentify new neurological, cardiovascular, haematopoiet-ic, ageing and immune phenotypes using high-through-put technologies.

Although mouse stocks can be quickly archived asfrozen sperm, the logistics of breeding, complementationtesting and mapping in the mouse may prove limiting.Furthermore, characterizing the pathology of mousemutants to make them relevant to human disease can bea daunting task. Recognizing this fact, the NIH has estab-lished a Mouse Genomics and Genetics ResourcesCommittee to suggest and implement strategies forimproving the infrastructure for mouse genetics27,28.These initiatives include training in pathobiology,improving genomic resources, improving sperm-freezingmethods, and establishing new mutant archiving and dis-tribution centres.

discarded, leaving only about 30% of the originalmutant collection. Still, the remaining collection ofover 2,000 mutations has been extremely valuable forstudying developmental processes in vertebrates, andfurther mutagenesis efforts using insertional mutagensare continuing24.

The current mouse mutagenesis efforts pale in com-parison with these model organisms (TABLE 2). However,the mouse is a complex mammal, and unique recessivephenotypes will probably be discovered more slowly,even though many represent disease phenotypes. Forexample, the mouse progressive ankylosis mutation(ank) causes a generalized, incremental form of arthritis,accompanied by mineral deposition, bony outgrowthsand joint destruction25. The ank gene encodes a multipass transmembrane protein that controlspyrophosphate levels, providing functional insight intothe development of arthritis26. Although the ank gene ishighly conserved in vertebrates, including zebrafish, rats,mice, cows and humans, it is not found in bacteria, yeast,worms or flies. Mutations affecting skeletal developmentand maintenance can be easily seen in the mouse as adysmorphology or an abnormal gait, and many will havedirect relevance to debilitating human diseases.

In any ENU screen, mutation isolation relies on thephenotypic assay, requiring that the mutant phenotypemust vary significantly from the background. In mousescreens, there should be an emphasis on assays for clinicalphenotypes that model human diseases; clinical testing ofmouse blood, for example, can yield a considerable arrayof phenotypes relevant to human disease. A completeblood count (CBC) with microscopic differential analysiscan identify abnormalities in red blood cell and whiteblood cell numbers or morphology, as well as plateletabnormalities. Extending the analysis of blood cells withflow cytometry may uncover other immunologicaldefects, as was carried out in the GSF screen. Clinicalchemistry tests can diagnose several organ system anom-alies, including liver, pancreatic, heart and kidney disor-ders. Urine analysis on mice can reveal increased levels ofglucose or other abnormal by-products of disease. High-throughput biochemical analysis, such as tandem mass

Chromosome 11

Trp53 24% recombination Wnt3

Inversion

Wnt3 0% recombination Trp53

a

b

Figure 3 | A mouse balancer chromosome. a | The firstbalancer chromosome was engineered to disrupt Trp53 andWnt3 on chromosome 11, which causes embryos that arehomozygous for the balancer to die during embryonicdevelopment. This balancer suppresses recombination over1/3 of the chromosome, is dominantly marked with theyellow coat conferred by the K14-agouti transgene, and islethal when homozygous. b | A wild-type mouse (right) with amouse heterozygous for the balancer chromosome (left).Note that the mice are not completely yellow, but have ayellowish ventrum, as well as yellow ears and tail (not shown).

Table 2 | Large-scale chemical mutagenesis in model organisms

Drosophila Zebrafish Mouse

Chemical EMS ENU ENU

Mutants isolated >20,000 >6,500 >1,000

Mutants confirmed ~600 >2,000 >300

Class Recessive Recessive Dominant

Advantages Genetic reagents Non-uterine Comparative linkage mapdevelopment

Generation time Parthenogenesis Clinically relevant phenotypes

Maintenance Ease of rearing Sperm freezing

Sperm freezing

The fly screens represent saturation mutagenesis of three chromosomes: X, second and third.‘Mutants isolated’ represents the number of lethal mutations, and the mutants confirmed werethose that died before hatching and had patterning defects. Numerous mutations were isolated in zebrafish screens; however, only about 2,000 lines were maintained as stocks.



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wide (FIG. 2), incorporating specialized phenotypicscreens for immune defects29.

Balancer chromosomes: isolating recessive mutations. Asecond approach to isolating recessive-lethal and detri-mental mutations is being carried out by this authorand Allan Bradley at Baylor College of Medicine, USA(Baylor mouse genome project). Using Cre/loxP chro-mosome engineering, inversions are being engineeredon individual mouse chromosomes. To make these bal-ancer chromosomes (BOX 2) similar to those that areconstructed in flies, loxP sites are placed in opposite ori-entations at two different locations on a mouse chromosome in ES cells. On the introduction of Crerecombinase, a recombination event produces aninverted chromosomal rearrangement. One end of theinversion is targeted with a vector that contains the K14-agouti transgene. This construct expresses the agoutigene under the control of the skin-specific promoter,keratin 14 (REFS 30,31). The first balancer chromosomehas been engineered on chromosome 11, in which oneendpoint in the first inversion breaks in Wnt3, a genethat is essential for early embryonic development (FIG. 3a). Disruption of this gene in mice homozygous forthe balancer chromosome causes them to die duringembryogenesis. The balancer chromosomes are used inschemes that are designed to isolate lethal or detrimen-tal mutations by screening for the presence or absenceof the coat-colour phenotype that they confer (FIG. 3b).This means that the mutations that are linked to the bal-ancer can be easily scored in a few crosses, eliminatingthe need to map them. Finally, the balancers are usefulfor the long-term maintenance and characterization ofthe new mutations. Because the mutations are isolatedin a three-generation pedigree mating scheme (FIG. 4),recessive lethal and visible mutations linked to the bal-ancer, and which are therefore mapping on the chromo-some of interest, are isolated at a high frequency.However, recessive viable mutations located elsewherein the genome are also observed, and need to bemapped in the traditional way.

Gene-based screens. An alternative approach to screeningfor desired phenotypes is to screen for DNA lesions in agene of interest and to determine a subsequent pheno-typic effect. Two different groups, using ENU and EMS,have shown that this approach can be effectively carriedout in mouse ES cells32,33. ES cells are treated with thechemical in culture, and the genes of interest are screenedfor point mutations. Alternatively, the cultured cells canbe screened for a phenotype. Any ES cell that contains adesired mutation can be clonally expanded and used togenerate chimeric mice that can transmit the mutation inthe germ line. This may be the most powerful approachfor saturating a single gene or locus with mutations.These studies have the added benefit that they will deter-mine which changes in the gene are neutral and fail tocause any noticeable phenotype in the whole organism.Using this strategy, identifying the DNA lesions in thewhole organism as well as in ES cells should be robustand cost-effective. Further technological developments

Future approaches to mouse mutagenesisIsolating recessive mutations genome-wide. Several newmutagenesis centres are being established in the UnitedStates through cooperative agreements with the NIH,and have already been established in Australia, Japanand Canada. To identify novel loci and further definegene function, some of these groups are beginning thelarge-scale isolation of recessive mutations usinggenome-wide and regional screens. A genome-widestrategy for recessive mutations requires a three-genera-tion breeding scheme (FIG. 2). This strategy is being car-ried out in parallel to the dominant screen by the GSFconsortium, which is planning to screen 800 mutage-nized genomes for recessive mutations. Similarly, ChrisGoodnow at Australian National University in Canberrahas focused on isolating recessive mutations genome-

a ENU

X

bX

c

X

d

G0

G2

G1

G2

Mutant class Carrier Dies

G2

G3:

G2

Figure 4 | Isolating recessive detrimental mutations with a marked balancerchromosome. This approach is being used for the saturation mutagenesis of mousechromosome 11 (REF. 4). In this scheme, an inversion generated by Cre/loxP is tagged with aK14-agouti transgene to produce mice with a dominant yellow coat. Black coat colouredC57BL/6J males are treated with a 3 x 100 mg kg–1 dose of ENU and are allowed to recoverfertility (G0). a | Fertile mutagenized males are mated to females carrying a balancerchromosome (shown as a double-headed arrow) to generate G1 animals that may carry a newmutation (shown in red). b | G1 animals (males or females) are mated to animals carrying botha balancer chromosome and a marker for the non-mutagenized chromosome (such as theRex allele, which gives mice a dominant curly coat, as shown by the curly lines). c | InformativeG2 animals are identified by their yellow coat colour and are intercrossed to look for newmutations in the G3 test class of offspring. d | Any pedigrees with greater than 24 offspring andno black animals must carry a new lethal mutation on chromosome 11. Lethal or detrimentalmutations can be rescued by the yellow-marked carriers. Mutations are mapped if theysegregate in trans to the yellow coat-colour marker. Visible mutations mapping genome-widemay also be isolated if they are detected in both test and carrier offspring.



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databases, which are being developed using standard-ized phenotype vocabularies to make descriptions andsearch terms uniform (The gene ontology project)34.Community cooperation is required to manage and dis-tribute stocks, characterize phenotypes and functionallyannotate the genome.

The addition of a complex mammal, such as themouse, to the list of informative genetic model organ-isms will allow an analysis of conserved and divergentevolution among species. ENU mutagenesis could alsobe used to dissect physiological parameters in othercomplex mammals, such as the rat, for which pilotscreens are being considered. Further mutagenesisefforts in mammals are likely to produce phenotypesthat will have an enormous effect on drug developmentand human health.

from the Human Genome Project may also facilitatemutation detection, including DNA sequencing byhybridization to DNA chips, and high-efficiency singlenucleotide polymorphism (SNP) detection.

Mutagenesis and the community. A large number ofENU-induced mutations have already been producedthat represent only a fraction of the potential of themammalian genome, and further mutagenesis experi-ments will generate new human disease models andreveal new mammalian developmental pathways. Inaddition to the programmes reported here, several newmutagenesis centres are being established in the US,Japan and Canada28. The mutants will soon be availableto numerous smaller laboratories, whose specialization,for example in physiology or endocrinology, will enablefurther dissection of the biological outcome of novelmutations. The mutagenesis centres could also aidsmaller laboratories by providing ENU-treated males ortheir G1 offspring for smaller-scale experiments.Furthermore, mutagenesis centres could act as hosts tovisiting scientists from more specialized laboratories.These scientists could help to develop new phenotypeassays to isolate their favourite mutants, which couldthen be taken away for further investigation.

The large-scale comparative analysis of mouse andhuman diseases requires the generation of phenotype

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AcknowledgementsThe author thanks H. Bellen for the critical reading of this manuscriptand for sharing his knowledge of the history and current status ofDrosophila melanogaster genetics, and Andrew Sallinger for pho-tographing the mice in FIG. 3. This work was supported by a UnitedStates Public Health Service grant.

Links

DATABASE LINKS Kit | Kitl | Whl | ank | Wnt3FURTHER INFORMATION UK ENU mutagenesisprogramme | German ENU-mouse mutagenesis project |Drosophila stock centre | Mouse Genomics and GeneticsResources Committee | Australian mutagenesis centre |Canadian mutagenesis centre | Chris Goodknow atAustralian National University | Baylor Mouse GenomeProject | The Gene Ontology Project | Mouse GenomeDatabase (MGD)


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