Large-scale mutational analysis for the annotation of the mouse genome

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17 After sequencing the human and mouse genomes, the annotation of these sequences with biological functions is an important challenge in genomic research. A major tool to analyse gene function on the organismal level is the analysis of mutant phenotypes. Because of its genetic and physiological similarity to man, the mouse has become the model organism of choice for the study of genetic diseases. In addition, there is at the moment no other vertebrate for which versatile techniques to manipulate the genome are as well developed. Several mouse mutagenesis projects have provided the proof- of-principle that a systematic and comprehensive mutagenesis of every gene in the mammalian genome will be feasible. An exhaustive functional annotation of the mammalian genome can only be achieved in a combination of phenotype- and gene- driven approaches in large- and small-scale academic and private projects. Major challenges will be to develop standardised phenotyping protocols for the clinical and pathological characterisation of mouse mutants, the improvement of mutation detection methods and the dissemination of resources and data. Beyond gene annotation, it will be necessary to understand how gene functions are integrated into the complex network of regulatory interactions in the cell. Addresses Institute of Experimental Genetics, GSF – National Research Center for Environment and Health, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany *e-mail: [email protected] Current Opinion in Chemical Biology 2001, 6:17–23 1367-5931/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Published online 29 November 2001 Abbreviations ENU N-ethyl-N-nitrosourea ES cells embryonic stem cells From genetics to genomics A paradigm shift is currently taking place in the life sciences. Biological processes are being analysed more and more by large scale and high-throughput approaches. The international Human Genome Project reflects this devel- opment very well. Instead of sequencing individual gene loci, the entire human genome is being sequenced in this project. Instead of looking at the expression of one specific gene during development or at a particular physiological state, strategies have been developed to monitor expression of thousands of genes in a single experiment. Such systematic approaches were pioneered by those researchers who successfully implemented large-scale approaches, for example, for the construction of a first genetic map of the Drosophila genome by TH Morgan or for the systematic mutagenesis of the genomes of Drosophila [1,2], Caenorhabditis [3,4], Arabidopsis [5] and Danio [6,7]. These projects have been of tremendous value for the understanding of the function of genes within the respective organisms. The generated mutant resources were the basis for the fundamental insight that genetic pathways have been remarkably conserved during evolu- tion. The multiple deployment of these pathways (or networks) for different functions is rather the rule than the exception [8–10]. The recent years of human genome research have been characterized by candidate gene approaches and positional cloning to identify monogenic human-disease genes, such as muscular dystrophy [11,12], Huntington’s disease [13,14] and cystic fibrosis [15,16]. At the same time, tools have been developed to handle, for example, high numbers of clones, sequences and hybridizations. This development was accompanied by the formation of a new biotech industry in genomics. A draft version of the human genome sequence has been established [17,18 •• ,19 •• ] and a large fraction of human genes has been mapped to their chromosomal region. The precision of such genetic maps is continuously improving (for a review, see [20 •• ]). A major focus of genomic research now is the systematic analysis of gene function. The anno- tation of gene maps with phenotypic information will add meaning and interpretation to the genomic sequence. This knowledge will help improve understanding of the molecu- lar basis and genetics of the pathobiology of human diseases. From mouse mutants to gene function A major tool for the study of gene function on the organismal level is the analysis of mutation phenotypes. The mouse is the most important model organism for human genetic disease and mammalian developmental genetics [21 •• ]. In its physiology and development, the mouse is very similar to humans. This is exemplified by many mutations that were identified in the mouse that cause phenotypes which are remarkably similar to the clinical symptoms of the corresponding human genetic diseases. In these cases, the mouse can serve as a model organism that may be used to understand the molecular mechanism of the disease, to identify surrogate markers that support the early diagnosis of the associated disease, and to find new molecular targets for therapy. Extensive comparative linkage maps that are already available for mouse and man also support the usefulness of the mouse as the major model organism for human genetic diseases. Such syntenic regions contain homologous genes in a Large-scale mutational analysis for the annotation of the mouse genome Johannes Beckers* and Martin Hrabé de Angelis

Transcript of Large-scale mutational analysis for the annotation of the mouse genome

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After sequencing the human and mouse genomes, theannotation of these sequences with biological functions is animportant challenge in genomic research. A major tool toanalyse gene function on the organismal level is the analysis ofmutant phenotypes. Because of its genetic and physiologicalsimilarity to man, the mouse has become the model organismof choice for the study of genetic diseases. In addition, there isat the moment no other vertebrate for which versatiletechniques to manipulate the genome are as well developed.Several mouse mutagenesis projects have provided the proof-of-principle that a systematic and comprehensive mutagenesisof every gene in the mammalian genome will be feasible. Anexhaustive functional annotation of the mammalian genome canonly be achieved in a combination of phenotype- and gene-driven approaches in large- and small-scale academic andprivate projects. Major challenges will be to developstandardised phenotyping protocols for the clinical andpathological characterisation of mouse mutants, theimprovement of mutation detection methods and thedissemination of resources and data. Beyond gene annotation,it will be necessary to understand how gene functions areintegrated into the complex network of regulatory interactionsin the cell.

AddressesInstitute of Experimental Genetics, GSF – National ResearchCenter for Environment and Health, Ingolstaedter Landstrasse 1,85764 Neuherberg, Germany*e-mail: [email protected]

Current Opinion in Chemical Biology 2001, 6:17–23

1367-5931/01/$ — see front matter© 2001 Elsevier Science Ltd. All rights reserved.

Published online 29 November 2001

AbbreviationsENU N-ethyl-N-nitrosoureaES cells embryonic stem cells

From genetics to genomicsA paradigm shift is currently taking place in the life sciences. Biological processes are being analysed more andmore by large scale and high-throughput approaches. Theinternational Human Genome Project reflects this devel-opment very well. Instead of sequencing individual geneloci, the entire human genome is being sequenced in thisproject. Instead of looking at the expression of one specificgene during development or at a particular physiologicalstate, strategies have been developed to monitor expression of thousands of genes in a single experiment.Such systematic approaches were pioneered by thoseresearchers who successfully implemented large-scaleapproaches, for example, for the construction of a first

genetic map of the Drosophila genome by TH Morgan orfor the systematic mutagenesis of the genomes ofDrosophila [1,2], Caenorhabditis [3,4], Arabidopsis [5] andDanio [6,7]. These projects have been of tremendous valuefor the understanding of the function of genes within therespective organisms. The generated mutant resourceswere the basis for the fundamental insight that geneticpathways have been remarkably conserved during evolu-tion. The multiple deployment of these pathways (ornetworks) for different functions is rather the rule than theexception [8–10].

The recent years of human genome research have beencharacterized by candidate gene approaches and positionalcloning to identify monogenic human-disease genes, such as muscular dystrophy [11,12], Huntington’s disease[13,14] and cystic fibrosis [15,16]. At the same time, tools have been developed to handle, for example, high numbers of clones, sequences and hybridizations. Thisdevelopment was accompanied by the formation of a newbiotech industry in genomics.

A draft version of the human genome sequence has beenestablished [17,18••,19••] and a large fraction of humangenes has been mapped to their chromosomal region. Theprecision of such genetic maps is continuously improving(for a review, see [20••]). A major focus of genomic researchnow is the systematic analysis of gene function. The anno-tation of gene maps with phenotypic information will addmeaning and interpretation to the genomic sequence. Thisknowledge will help improve understanding of the molecu-lar basis and genetics of the pathobiology of human diseases.

From mouse mutants to gene functionA major tool for the study of gene function on the organismal level is the analysis of mutation phenotypes.The mouse is the most important model organism forhuman genetic disease and mammalian developmentalgenetics [21••]. In its physiology and development, themouse is very similar to humans. This is exemplified bymany mutations that were identified in the mouse thatcause phenotypes which are remarkably similar to the clinical symptoms of the corresponding human genetic diseases. In these cases, the mouse can serve as a modelorganism that may be used to understand the molecularmechanism of the disease, to identify surrogate markersthat support the early diagnosis of the associated disease,and to find new molecular targets for therapy. Extensivecomparative linkage maps that are already available formouse and man also support the usefulness of the mouseas the major model organism for human genetic diseases.Such syntenic regions contain homologous genes in a

Large-scale mutational analysis for the annotation of themouse genomeJohannes Beckers* and Martin Hrabé de Angelis

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similar spatial order along chromosomal regions. This simplifies candidate gene approaches because phenotypicannotation that may exist for the model organism may hintto the affected genes in the critical regions associated witha genetic human disease, and vice versa.

An important prerequisite for the consistent phenotypicanalysis of mutant alleles is that gene function can beanalysed on defined and stable genetic backgrounds.Working with inbred genetic backgrounds simplifies theanalysis of mutant phenotypes [22]. Because inbredstrains are principally homozygous for any given geneticlocus, phenotypic variations between individual mice arereduced to a minimum. Approximately 100 laboratorymouse strains and many times this amount of sub-strains,each with a characteristic set of phenotypic traits, are

currently used for functional genomics in the mouse. Inaddition, the phenotypic variation between such inbredmouse lines is a rich source for natural genetic variants. Aworld-wide community effort is currently ongoing to systematically describe strain-specific differences covering,for example, physiological, immunological, anatomical,neurobiological, behavioural and other parameters(www.jax.org/phenome; see Table 1 for a list of URLs).The majority of phenotypic characteristics that distin-guish mice of different strains are so called quantitativetraits. These vary in a continuous manner in their expres-sion and can be due to genetic as well as environmentalfactors, or a combination of both. Just as for genes responsible for the expression of qualitative traits, genesinvolved in the expression of continuous traits can bemapped by crossing inbred mouse strains, provided a

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Table 1

Collection of important internet links to mouse mutagenesis, gene trap, and sequencing centres.

Instutions Description URLInternational Mammalian Genome Society General http://www.imgs.org/European Mouse Mutant Archive Mutant archive http://corba.ebi.ac.uk/EMMA/The Jackson Laboratory, Maine, USA Mutant archive http://www.jax.org/resources/documents/GSF – National Research Centre, Munich, ENU mutagenesis http://www.gsf.de/ieg/groups/enu-mouse.html GermanyMammalian Genetics Unit, Harwell, UK ENU mutagenesis http://www.mgu.har.mrc.ac.uk/mutabase/Howard Hughes Medical Institute, ENU mutagenesis http://www.northwestern.edu/neurobiology/faculty/takahashi.html Northwestern University, IL, USAMcLaughlin Research Institute for ENU mutagenesis http://www.montana.edu/wwwmri/enump.html Biomedical Sciences, Montana, USAMedical Genome Centre, Canberra City, ENU mutagenesis http://jcsmr.anu.edu.au/group_pages/mgc/mutagenesis/mutagenesis.html AustraliaBaylor College of Medicine, Texas, USA ENU mutagenesis http://www.mouse-genome.bcm.tmc.edu/ENU/ENUhome.aspTennessee Mouse Genome Consortium, ENU mutagenesis http://tnmouse.org/ USARIKEN, Institute of Physical and Chemical ENU mutagenesis http://www.gsc.riken.go.jp/e/Mouse/index.htm Research, JapanThe Jackson Laboratory, Maine, USA ENU mutagenesis http://www.jax.org/hlbs/index.html

http://www.jax.org/nmf/Samuel Lunenfeld Research Institute, ENU mutagenesis, http://www.cmhd.ca/ Toronto, Canada Gene trapBay Genomics Consortium, USA Gene trap http://baygenomics.ucsf.edu/Manitoba Institute of Cell Biology, Canada Gene trap http://www.umanitoba.ca/institutes/manitoba_institute_cell_biology/MICB/hicks_

geoff.htmThe German Gene Trap Consortium, GSF Gene trap http://tikus.gsf.de/ – National Research Centre, Munich GermanyUniversity of California Gene Trap Gene trap http://ist-socrates.berkeley.edu/~skarnes/resource.html ResourceGenome Sequence Centre, Canada BAC map http://www.bcgsc.bc.ca/projects/mouse_mapping/Genome Sequencing Center, Washington BAC map http://genome.wustl.edu/gsc/mouse/ University, MO, USANational Center for Biotechnology Trace Repository http://www.ncbi.nlm.nih.gov/Traces/ InformationEnsemble Mouse Genome Server Mouse/human

genomehttp://mouse.ensembl.org/

comparisonUCSC, Human Genome Project Working Mouse/human http://genome.cse.ucsc.edu/ Draft genome

comparisonThe Jackson Laboratory, Maine, USA Genes, markers, http://www.informatics.jax.org/searches/marker_form.shtml

phenotypesThe Jackson Laboratory, Maine, USA Mouse phenotype http://aretha.jax.org/pub-cgi/phenome/mpdcgi?rtn=docs/home

databaseSamuel Lunenfeld Research Institute, Cre transgenic http://www.mshri.on.ca/nagy/cre.htm Toronto, Canada database

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reliable screen to measure the quantitative trait is established. The characterization of quantitative trait lociis particularly relevant for the identification of humanalleles involved in disease predisposition.

Despite a century of mouse genetic research, less than 5000out of an expected total of 30 000 genes have functionsattributed to them on the basis of direct experimental stud-ies. Based on sequence analysis, no prediction of functioncan be made for as many as 30% of the genes of the humangenome, and the inferred functions of most of the remain-ing genes have yet to be proven. A comprehensivefunctional annotation of the mammalian genome can onlybe achieved in a combination of phenotype- and gene-driven approaches in large-scale and small academic andprivate projects. To this end, an international consortiumhas been brought together to systematically and compre-hensively assign functions to every gene in the genome andto identify every gene that affects traits of high biomedicalinterest [23••]. Here, we give an overview of the technolo-gies and methods of choice that provide the basis for a firstdraft of a comprehensive functional annotation map.

Gene-driven approachesThere is currently no other vertebrate than the mouse forwhich such versatile techniques to manipulate the genomeare sufficiently well developed. Conceptually, these muta-genesis approaches may be distinguished as so-called gene-and phenotype-driven technologies. Principally, the formerapproach starts with a known gene that is intentionallymodified and subsequently analysed for phenotypic varia-tions, which then are indicative of a gene function at theorganismal level, whereas the latter begins with an unchar-acterised mutation and an existing phenotype (a disease, abehavioural difference, a morphological change, etc.).

A major tool for the gene-driven approach is based onmouse embryonic stem cells (ES cells) [24]. These cellsare pluripotent cells that, in particular, have maintainedthe capacity to contribute to the male germ line (ES cellsare generally derived from male mice). ES cells are main-tained and propagated in cell culture under conditions thatprevent them from differentiation. By electroporation, targeting constructs typically containing two arms homo-logous to the gene to be modified, a desired modificationof the gene (mutation), and a selectable marker are intro-duced into the cell. Based on DNA repair mechanisms thatare intrinsic to the cell, the targeting vector integrates(with a sufficiently high frequency) at the site of homologyby homologous recombination. Once clones with thedesired mutation have been identified, they are injectedinto mouse blastocysts to generate chimeras that transmitthe mutated allele through the germ line. Such homolo-gous recombination technology in mouse embryonic stemcells allows the deletion or integration of chunks of DNAat almost any locus in the genome, provided the sequenceof the gene to be targeted is known [25,26]. For severalyears now, tools have been used to generate mutations

such that selectable markers can be removed using theyeast recombinase Cre to avoid unwanted influences ofthese ectopic sequences on the mutated or neighbouringgenes [27]. During meiosis, the same recombination mechanism may also be used to delete or duplicate largegenomic regions or to transfer alleles in trans to closelylinked alleles in cis in an application called trans-allelic targeted meiotic recombination [28]. Beyond that, mutationsintroduced by homologous recombination may also bedesigned such that they are inducible, for example, at aparticular developmental stage, in specific tissues or by anartificial activator [29–31].

Another important genetic tool for the study of gene function and gene regulation is and has been the transgenicmouse system. In this case, a DNA construct, typicallycontaining a promotor and regulatory unit and a gene ofinterest or a reporter gene, is injected in one of the pro-nuclei of the fertilised egg [32]. The DNA generallyintegrates in multiple copies at a random position into thegenome. Classically, such transgenes are used to study theeffects of regulated over-expression of a functional geneproduct. In addition, the transgenic mouse system has suc-cessfully and frequently been used to study the functionalpotentials of regulatory DNA sequences. Both transgenesisand homologous recombination have also been used tostudy gene function across species. Although the trans-genic mouse system is faster than the ES cell technique, amajor drawback has been that the integration site influences the expression of transgenes. However, tech-niques have been developed to reduce these effects by co-injecting DNA sequences that insulate the transgene fromsurrounding regulatory influences [33–35].

Both technologies, gene-targeting in ES cells and thetransgenic mouse system, require that the gene to bemanipulated has been isolated and that the structure of thegene is known, at least partially. These approaches werefundamental for the functional analysis of specific molecularpathways in a mammalian organism. The majority of the molecular pathways that have been investigated were previously known from functional studies in other organisms such as the fly or yeast.

Another gene-driven approach that also allows the mutagenesis of uncharacterised genes is the gene-traptechnology [36]. In this strategy, ES cells are electroporatedwith a so-called gene trap vector. These vectors aredesigned to integrate at random sites in the genome and touse the transcriptional activity of the endogenous targetgene to drive a selection cassette and eventually a reportergene. The integration of the gene trap vector may gener-ate an insertional mutation. The gene trap vector alsoprovides a tag to easily identify the targeted gene and thereporter gene may be used for an expression analysis, forexample, for the identification of genes with specificexpression patterns during embryogenesis [24]. Mice gen-erated from gene-trapped ES cells are subsequently used

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for the phenotypic analysis. The gene-trap technology is apowerful tool for combining large-scale random mutagene-sis in the mouse with a rapid identification of the mutatedgene [37].

The challenge with such gene-based methods is that thephenotypic consequences of the generated mutations maybe unexpected or difficult to detect. Standardised tech-nologies for a wide range of phenotypic parameters areurgently needed and are a major goal of the InternationalMouse Mutagenesis Consortium [23••]. Some of the majorchallenges are, for example, to establish a wide range ofstandardised phenotypic assays with defined protocolsand conditions, and to put in place efficient networks for the exchange of information and resources. These infrastructures will, of course, be beneficial also for phenotype-driven approaches.

Phenotype-driven approachesThe first mutant mice were isolated more than a centuryago, based on curious phenotypes, by animal breeders.Many coat colour, circling, or kinky tail mice were discov-ered in breeding colonies, because their abnormalphenotype could easily be detected. This has led to a col-lection of more than 1200 mutants. Because of the randomnature and the rare occurrence of such natural mutations,there has been an interest in developing techniques toinduce mutations. In 1927, Muller [38] drew the conclusionthat heritable mutations can be induced by exposure toX-rays. Since then, several groups investigated gene func-tion using radiation as a mutagen. Subsequently, variouschemicals were used and tested as mutagenic substances.Initially, these agents were assessed for their risk for humanhealth using mice as model system. At the same time, novelalleles of genes were generated that could be used for theanalysis of gene function. N-ethyl-N-nitrosourea (ENU),was found to be one of the most powerful mutagens for theproduction of mouse mutants [39–41]. In contrast to X-rays,ENU mainly introduces point mutations and efficientlymutagenizes mouse premeiotic spermatogonial cells, suchthat large numbers of mutant mice can be obtained fromfew ENU-treated male mice. Because of the nature ofENU-induced mutations, phenotypes range from hypo-morphic and loss-of-function alleles to hypermorphic anddominant negative alleles [42]. As mentioned above, sys-tematic ENU mutagenesis screens have successfully beenperformed in several non-mammalian organisms. The firstENU mutagenesis screens that were carried out in micefocused on specific chromosomal regions or phenotypes[43–46]. As an example, one of the largest genome-widescreens for a specific phenotype identified dominant muta-tions causing cataracts in mice [47]. More than 500 000mice were screened for eye lens opacity with an ophtalmo-logical device, the slit lamp. 170 mutants were isolated inthis screen. The chromosomal localisation of several muta-tions has now been mapped. In some cases, more than 10alleles of the same locus were recovered; in many, a few oronly one allele has been found.

Nevertheless, today there are still far fewer mouse mutantsavailable than there are genes and, even more so, thanthere are informative alleles — a phenomenon that hasbeen termed ‘the phenotype gap’ [48]. To narrow this gap,some recent projects have successfully focused on large-scale and systematic ENU mutagenesis approaches toisolate new mutants and allelic series that are particularlyrelevant for human genetic diseases [49]. To this end,assays that are routinely used in clinical diagnosis havebeen adopted for the mouse and new protocols have beendeveloped for the isolation of novel neuropathological andbehavioural phenotypes. Such projects were first initiatedat the MRC, Harwell, United Kingdom and at the GSFResearch Centre in collaboration with the LMU Munich,Germany [50••,51••]. In these screens, protocols weredeveloped to assess mutant phenotypes, for example, forspecific, prenatal and postnatal abnormalities, comprisingcongenital malformations, clinical chemical, biochemical,haematological, immunological parameters and complextraits such as allergy and behavior (for phenotype assayssee [52]). Up to now, more than 500 new genetically con-firmed mouse mutant lines have been established in theseprojects. Routinely, each mutation is being mapped usinga panel of polymorphic markers at approximately 10 cMintervals. Using the candidate gene approach, severalgenes and their mutations have already been identified(e.g. [53,54]). Similar genome-wide ENU mutagenesisscreens are currently being implemented at institutions inAustralia, Japan, Canada, the USA and other countries (seeTable 1 for internet links to these institutions).

An alternative to such genome-wide scans is a region-specific scan. Mating, for example, ENU injected males tofemales carrying a deletion at a chromosomal region ofinterest leads to homozygous animals for the effected genewithin that region in the F1 generation. In this case, thephenotype is the product of two different alleles, namelythe point mutation derived from the father and the nullmutation (deletion) obtained from the mother. In a pioneering project using this strategy, the albino deletioncomplex was saturated with ENU-induced mutations. Arich collection of mutants was identified, among themmany with medically relevant phenotypes. The clearadvantage of the region-specific scan is the high number ofmice that can be analyzed for recessive mutations at aknown chromosomal position [55,56]. The availability ofwell-defined deletions is a limiting factor for region-specificmutagenesis screens. The application of site-specificrecombinases such as the loxP/Cre-recombinase system willbe one way to provide genotypes with precisely definedseries of genomic deletions. However, the GSF/LMU andMRC large-scale mutagenesis screens have demonstratedthat new recessive alleles can efficiently be isolated alsofrom unbiased, genome-wide screens.

So called sensitised mutagenesis screens provide a meansfor the identification of new alleles associated with partic-ular functions. Generally, ENU-mutagenised male mice

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are crossed to a female carrying either a mutation in a geneof interest or a transgenic female that is, for example, a disease model. The goal of such screens is to identify newalleles that modify the phenotype of the original mutationor the transgenic model in one way or another. Thus, sen-sitised screens are particularly useful tools for the isolationof new alleles of known or unknown genes that areinvolved in particular biological processes or that are closelyassociated with the molecular pathway of the originalmutation that was used in the sensitised screen.

Having at hand these diverse techniques to manipulate themouse genome using gene-driven as well as phenotype-driven approaches it appears feasible, for the first time, tosystematically and comprehensively analyse gene functionin a mammal closely related to man. Besides the establish-ment of new diverse and standardised phenotypingprotocols, a major challenge in this undertaking is thedevelopment of new technologies, for example, for map-ping mutations and genotyping. Several projects areongoing to develop high-throughput genotyping methodsthat do not require gel-electrophoresis and that are basedon single nucleotide polymorphisms (SNPs) of inbredmouse strains [57]. The establishment of SNP maps forsets of different mouse strains will very much simplify theautomation of genetic linkage analyses. A project to comprehensively annotate the genome with at least onefunction for every gene can only be achieved in a commu-nity effort. To reach this objective, the InternationalMouse Mutagenesis Consortium (IMMC) has proposedseveral long-range goals and has defined some of the keychallenges for a comprehensive mouse functionalgenomics project [23••]. Networks of laboratories are cur-rently being established for the distribution of resourcesand information.

Gene regulation, the next levelBeyond the annotation of the genome with functions forevery gene, we will also have to try to understand howthese gene functions are integrated into the complex network of molecular interactions of the cell. Most biochemical processes within and between cells are putinto effect by the interaction between proteins, or betweenproteins and their substrates. The proteome of a cell is theresult of controlled biosynthesis, and hence largely (butnot exclusively) regulated by gene expression. In turn,gene expression can be regarded as a sensitive read-out ofthe biochemical state of the cell, or in other words the pro-teome. In this regard, the genome (the set of genes perorganism) is merely the tool case of the living organism.When and where these tools are applied during the con-struction of the organism (the unfolding of the organismalBauplan), in homeostasis and disease is at the heart of afunctional and healthy organism. The ‘when and where’,the logistics, is primarily controlled by gene regulation.Transcriptome and proteome feedback to each other in ahighly complex and somehow controlled way. The under-standing of this functional regulation is very incomplete

and limited to a few isolated signalling or metabolic pathways. However, evidence is accumulating that suchpathways are in fact mere components of complicated networks ‘that integrate many inputs to generate the complex output that is cell behaviour’ [58].

DNA chips provide the technology to monitor genome-wide gene expression at the mRNA level (for review see[59]). New technologies are being developed to monitorproteomes at similar throughput rates. If it could beachieved to integrate techniques (such as laser microdis-section) to isolate homogenous populations of cells andtechniques for comparative transcriptome and proteomeanalysis, then it would be possible, for the first time, tocomprehensively analyse gene function in the context ofthe molecular network of the cell. Such a holistic approachof molecular analysis would have important synergisticeffects on the analysis of regulatory interdependencies thatdetermine the molecular phenotype of the cell; it wouldalso allow distinguishing between transcriptional and post-transcriptional regulation in a comprehensive approach.

Besides those new technologies to monitor the transcrip-tome and proteome, an important tool for the study of generegulatory mechanisms is the analysis of mutations withinregulatory sequences. Most of the regulatory mutants thathave been studied so far were generated by targeted muta-genesis in ES cells or by mutagenesis of transgenes. Veryfew regulatory mutants have been isolated and describedfrom undirected mutagenesis approaches. One reason forthis may be that the majority of point mutations in regula-tory elements, for example, induced by ENU mutagenesishave rather mild effects on the regulation of the associatedgene(s). In this sense, regulatory regions may be less sensitive to point mutations as compared with codingsequences. It remains to be shown whether regulatorymutations have a better chance to be discovered in recessive screens.

The mapping of mutations in phenotype-driven approachesis generally based on candidate gene approaches. Once thenumber of candidate genes has been reduced to a mini-mum, these genes are sequenced to detect the mutationwithin the coding region. At the moment, mutation detec-tion efforts are put to a halt in most cases if coding regionsare found to be normal. Thus, existing resources ofmutants in which the mutation detection via candidategene approach failed may represent a source of potentialregulatory mutants.

ConclusionsIn the next few years, large-scale and small-scale gene-driven and phenotype-driven mutagenesis projects willproduce high numbers of new mouse mutants. This willhelp build up mouse mutant resources of new alleles tocover a wide variety of human genetic diseases. The production of these mutants is a major challenge but notthe limiting step in functional genomics.

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In phenotype-driven mutagenesis approaches, cloning theresponsible gene and mutation has been time consumingand requires advanced genomics. The availability of com-plete genome sequences in combination with expressiondatabases, full-length cDNA libraries and new mutationdetection methods will make such cloning strategies muchmore efficient. To functionally analyse mutant alleles, afull clinical and pathological characterization of mutantmouse lines is necessary. A systematic and standardisedevaluation of phenotypes comparable to clinical examina-tions of human patients will be required. Enormousamounts of sequence and functional data will be generatedin the next years. The accessibility of this data for the scientific community will be an additional challenge. A significant part of resources for functional genomics willhave to be committed towards bioinformatics.

A genetic approach is key for the comprehensive analysis ofgene function. A combination of mouse mutagenesis andfunctional genomics will become the platform to geneticallydissect biological pathways. The mutant resources that aregenerated now will be the basic resources to exploit thepotential of genomics in the near future.

AcknowledgementsWe would like to thank Veronique Blanquet and colleagues from the lab forhelpful comments.

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