Functional genomics in the mouse: phenotype-based mutagenesis screens

Significant progress has been made in sequencing the genomes of several model organisms, and efforts are now underway to complete the sequencing of the human genome. In parallel with this effort, new approaches are being developed for the elucidation of the functional content of the human genome. The mouse will have an important role in this phase of the genome project as a model system. In this review we discuss and compare classical genetic approaches to gene function-phenotype-based mutagenesis screens aimed at the establishment of a large collection of single gene mutations affecting a wide range of phenotypic traits in the mouse. Whereas large scale genome-wide screens that are directed at the identification of all loci contributing to a specific phenotype may be impractical, region-specific saturation screens that provide mutations within a delimited chromosomal region are a feasible alternative. Region-specific screens in the mouse can be performed in only two generations by combining high-efficiency chemical mutagenesis with deletion complexes generated using embryonic stem (ES) cells. The ability to create and analyze deletion complexes rapidly, as well as to map novel chemically-induced mutations within these complexes, will facilitate systematic functional analysis of the mouse genome and corresponding gene sequences in humans. Furthermore, as the extent of the mouse genome sequencing effort is still uncertain, we underscore a necessity to direct sequencing efforts to those chromosomal regions that are targets for extensive mutagenesis screens.     

Comparative mapping of the human 22q11 chromosomal region and the orthologous region in mice reveals complex changes in gene organization

The region of human chromosome 22q11 is prone to rearrangements. The resulting chromosomal abnormalities are involved in Velo-cardio-facial and DiGeorge syndromes (VCFS and DGS) (deletions), "cat eye" syndrome (duplications), and certain types of tumors (translocations). As a prelude to the development of mouse models for VCFS/DGS by generating targeted deletions in the mouse genome, we examined the organization of genes from human chromosome 22q11 in the mouse. Using genetic linkage analysis and detailed physical mapping, we show that genes from a relatively small region of human 22q11 are distributed on three mouse chromosomes (MMU6, MMU10, and MMU16). Furthermore, although the region corresponding to about 2.5 megabases of the VCFS/DGS critical region is located on mouse chromosome 16, the relative organization of the region is quite different from that in humans. Our results show that the instability of the 22q11 region is not restricted to humans but may have been present throughout evolution. The results also underscore the importance of detailed comparative mapping of genes in mice and humans as a prerequisite for the development of mouse models of human diseases involving chromosomal rearrangements.     

Micronuclei in cytokinesis-blocked lymphocytes may predict patient response to radiotherapy

The recent explosion in the number of identified genes involved in the human skeletal dysplasias has dramatically advanced this particular field. While linkage efforts are mapping hereditary disorders of the skeleton at an ever accelerating pace, progress in the Human Genome Project is providing tools for rapid gene discovery after the map location is known. Emerging themes in the molecular analysis of the skeletal dysplasias include the identification of allelic series of disorders and the existence of mutational and genetic heterogeneity in many of these conditions. Allelic series include those conditions caused by mutations in the genes encoding type II collagen (COL2A1), cartilage oligomeric matrix protein (COMP), fibroblast growth factor receptor 3 (FGFR3) and the diastrophic dysplasia sulfate transporter (DTDST). The recognition of these phenomena has initiated the analysis of the relationship between disease phenotype and gene.     

The Human Genome Project: applications in the diagnosis and treatment of neurologic disease

The Human Genome Project (HGP), an international program to decode the entire DNA sequence of the human genome in 15 years, represents the largest biological experiment ever conducted. This set of information will contain the blueprint for the construction and operation of a human being. While the primary driving force behind the genome project is the potential to vastly expand the amount of genetic information available for biomedical research, the ramifications for other fields of study in biological research, the biotechnology and pharmaceutical industry, our understanding of evolution, effects on agriculture, and implications for bioethics are likely to be profound.     

Mouse mutagenesis-systematic studies of mammalian gene function

The mouse will play a pivotal role in mammalian gene function studies as we enter the post-genomics era. The challenge is to develop systematic, genome-wide mutagenesis approaches to the study of gene function. The current mouse mutant resource has been an important source of human genetic disease models. However, despite an apparently large catalogue of mouse mutations, we have access to mutations at only a small fraction of the likely total number of mammalian genes-there is a phenotype gap that needs to be filled by the establishment of new mutagenesis programmes. Two routes, genotype- and phenotype-driven, can be used for the recovery of novel mouse mutations. For the former, gene trap embryonic stem cell libraries appear set to deliver a large number of mutations around the mouse genome. The advantage of genotype-driven approaches is the ease of identification of the mutated locus; the disadvantage that a priori assumptions have to be made concerning the function and likely phenotype of the mutated gene. In contrast, phenotype-driven mutagenesis emphasizes the recovery of novel phenotypes. One phenotype-driven approach that will play an important role in expanding the mouse mutant resource employs the mutagen N-ethyl-N-nitrosourea (ENU). The phenotype-driven route makes no assumptions about the underlying genes involved, and ENU mutagenesis programmes can be expected to play a significant role in uncovering novel pathways and genes; the disadvantage is that the identification of the mutant gene is still not trivial. Together, the complementary routes of genotype- and phenotype-driven mutagenesis will provide a much enlarged catalogue of mouse mutations and phenotypes for future gene function studies.     

Disruption and sequence identification of 2,000 genes in mouse embryonic stem cells

The dramatic increase in sequence information in the form of expressed sequence tags (ESTs) and genomic sequence has created a 'gene function gap' with the identification of new genes far outpacing the rate at which their function can be identified. The ability to create mutations in embryonic stem (ES) cells on a large scale by tagged random mutagenesis provides a powerful approach for determining gene function in a mammalian system; this approach is well established in lower organisms. Here we describe a high-throughput mutagenesis method based on gene trapping that allows the automated identification of sequence tags from the mutated genes. This method traps and mutates genes regardless of their expression status in ES cells. To facilitate the study of gene function on a large scale, we are using these techniques to create a library of ES cells called Omnibank, from which sequence-tagged mutations in 2,000 genes are described.     

Identification of selected gamma-ray induced deficiencies in zebrafish using multiplex polymerase chain reaction

The ease with which mutations can be generated in zebrafish makes this vertebrate an important resource for developmental genetics and genome studies. We have developed a PCR-based screening method that allows the efficient identification of gamma-ray induced deficiencies targeted to selected sequences. We describe three mutants characteristic of our findings and show that these mutations include deletions and translocations that can affect as much as 1% of the genome. These deficiencies provide a basis for analyzing the functions of cloned zebrafish genes using noncomplementation screens for point mutations induced by high-efficiency chemical mutagenesis.     

Are genetic factors important in the aetiology of leukoaraiosis? Results from a memory clinic population

Although cumulative evidence suggests that a genetic predisposition plays a major role in development of systemic lupus erythematosus (SLE) and/or lupus nephritis (LN), the susceptibility genes are mostly unknown. The difficulty in identifying susceptibility genes is due in part to multiple genes with variable genetic effects and the diverse genetic backgrounds of human populations. In human SLE, genes of early components of complements as well as many polymorphic genes (including the MHC class II and class III, FcgammaR, mannose-binding protein, IL-6, Bcl-2, and IL-10 genes) have been associated with SLE or LN by population-based case-control or within-case studies. The contribution of some of these disease-associated genes to the presence or absence of clinical manifestations has been further tested in mice with targeted disruption of the specific candidate gene. In addition to SLE susceptibility genes, there may be a separate set of nephropathy susceptibility genes predisposing to LN as suggested by the familial clustering of end-stage renal disease in African-Americans with LN. The availability of densely mapped genetic markers spanning the entire genome has enabled the identification of chromosomal regions linked to disease susceptibility genes without prior knowledge of the gene function. Our group has used known murine lupus susceptibility loci as a guide, and conducted linkage analysis of genetic markers located within a specific, possibly syntenic human chromosomal region. Evidence for linkage of a chromosome 1q41-42 region was observed in SLE-affected sib pairs from multiple ethnic groups. More recently, several groups have reported results of genome scans of SLE-affected sib pairs or pedigrees. These exciting recent developments in delineating the genetic basis of SLE or LN are summarized in this review.     

Modulation of GDP-dissociation inhibitor protein membrane retention by the cellular redox state in adipocytes

Genetic screens in zebrafish have provided mutations in hundreds of genes with essential functions in the developing embryo. To investigate the possible uses of chromosomal rearrangements in the analysis of these mutations, we genetically characterized three gamma-ray induced alleles of cyclops (cyc), a gene required for development of midline structures. We show that cyc maps near one end of Linkage Group 12 (LG 12) and that this region is involved in a reciprocal translocation with LG 2 in one gamma-ray induced mutation, cyc(b213). The translocated segments together cover approximately 5% of the genetic map, and we show that this rearrangement is useful for mapping cloned genes that reside in the affected chromosomal regions. The other two alleles, cyc(b16) and cyc(b229), have deletions in the distal region of LG 12. Interestingly, both of these mutations suppress recombination between genetic markers in LG 12, including markers at a distance from the deletion. This observation raises the possibility that these deletions affect a site required for meiotic recombination on the LG 12 chromosome. The cyc(b16) and cyc(b229) mutations may be useful for balancing other lethal mutations located in the distal region of LG 12. These results show that chromosomal rearrangements can provide useful resources for mapping and genetic analyses in zebrafish.     

The Human Genome Project and the role of genetics in health care

The Human Genome Project, the mapping of our 100,000 genes and the sequencing of all of our DNA, will have major impact on biomedical research and the therapeutic and preventive health care. The tracing of genetic diseases to their molecular causes is rapidly expanding diagnostic and preventive options, while the increased insights into molecular pathways open tremendous perspectives for pharmacological and genetic therapies. The design of animal model systems for the functional study of disease and development of bioinformatics and biostatistics to improve our pattern recognition abilities are greatly accelerating progress. However, the optimal value from the current explosion of 'data mining' possibilities will only be gained when the basic data are made and kept publicly accessible, at the same time preventing the jeopardisation of the protection of intellectual property, arising from downstream inventions. This is one of the goals of HUGO, the international Human Genome Organisation, established 9 years ago to assist coordinating data acquisition and exchange and societal implementation of the genome project. Additional points of major importance in this historic endeavour are the safeguarding of a worldwide balance in the contribution and benefits to countries and population, the prevention of stigmatisation and discrimination of individuals and groups and the maintenance of respect for the priceless diversity of our world's cultures and traditions.     

Deletion mapping of four loci defined by N-ethyl-N-nitrosourea-induced postimplantation-lethal mutations within the pid-Hbb region of mouse chromosome 7

As part of a long-term effort to refine the physical and functional maps of the Fes-Hbb region of mouse chromosome 7, four loci [l(7)1Rn, l(7)2Rn, l(7)3Rn, l(7)4Rn] defined by N-ethyl-N-nitrosourea (ENU)-induced, prenatally lethal mutations were mapped by means of trans complementation crosses to mice carrying lethal deletions of the mouse chromosome-7 albino (c) locus. Each locus was assigned to a defined subregion of the deletion map at the distal end of the Fes-Hbb interval. Of particular use for this mapping were preimplantation-lethal deletions having distal breakpoints localized between pid and Omp. Hemizygosity or homozygosity for each of the ENU-induced lethals was found to arrest development after uterine implantation; the specific time of postimplantation death varied, and depended on both the mutation itself and on whether it was hemizygous or homozygous. Based on their map positions outside of and distal to deletions that cause death at preimplantation stages, these ENU-induced mutations identify loci, necessary for postimplantation development, that could not have been discovered by phenotypic analyses of mice homozygous for any albino deletion. The mapping of these loci to specific genetic intervals defined by deletion breakpoints suggests a number of positional-cloning strategies for the molecular isolation of these genes. Phenotypic and genetic analyses of these mutations should provide useful information on the functional composition of the corresponding segment of the human genome (perhaps human 11q13.5).     

Transgenic models in renal tubular physiology

Animal transgenesis has proven to be useful for physiological as well as physiopathological studies. Besides the classical approach based on the random integration of a DNA construct in the mouse genome, gene targeting can be achieved using totipotent embryonic stem (ES) cells for targeted transgenesis. Transgenic mice are then derived from the transgenic ES cells. This allows the introduction of null mutations in the genome (so-called knock-out) or the control of the transgene expression by the endogenous regulatory sequences of the gene of interest (so-called knock-in). Development of these transgenic animals leads to a better understanding of the cellular function of many genes or to the generation of animal models for human diseases. The purpose of this short review is to describe animal models in renal tubular physiopathology. Recent progresses will allow the generation of animal models with conditional expression of the transgene of interest or with a conditional gene mutation. This permits spatial and temporal control of the expression of the transgene or of the mutation. This should allow the generation of models suitable for physiological analysis or closer to disease state.     

Toward a cDNA map of the human genome

Advances in the Human Genome Project are shaping the strategies for identifying the 50,000-100,000 human genes. High-resolution genetic maps of the human genome combined with sequencing herald an era of rapid regional definition of disease genes. However, only once their chromosome band location is known will the systematic partial sequencing of thousands of random cDNA clones provide the reagents for teh rapid assessment of the genes responsible for the inherited disorders. We now present an approach to the rapid determination of map position and therefore to the creation of a transcribed map of the human genome. Sensitive fluorescence in situ hybridization has been combined with high-resolution chromosome banding and random cDNA sequencing to map 41 cDNAs with an average insert size of <2 kb to single human chromosome bands. The result provide 15 new genes, with database and functional information, as candidates for human disease. These include the large extracellular signal-related kinase (HUMERK), the ERK activator kinase (PRKMK1), a new member of the RAS oncogene family, protein phosphatase 2 regulatory subunit B alpha isoform (PPP2R2A), and a novel human gene with very high homology to a plant membrane transport family. Further, an analysis of expressed genes associated with pseudogenes showed that by using these techniques, it is possible to detect accurately the transcribed locus within a multigene or processed pseudogene family in most cases. These findings suggest that direct cDNA mapping using fluorescence in situ hybridization provides an accurate and rapid approach to the definition of a transcribed map of the human genome. This low-cost, high-resolution (2-5 Mb) mapping greatly enhances the speed with which these genes can be subsequently assigned to contigs. This assignment provides a necessary first step in understanding the relationship of the genes to both acquired and inherited human diseases.     

Polymerase chain reaction-mediated typing of microorganisms: tracking dissemination of genes and genomes

The polymerase chain reaction (PCR) is a powerful molecular biology tool which can be used for the identification of species and strains of diverse microorganisms. By aimed amplification of characteristic genes (i.e., genes encoding ribosomal RNA molecules) and subsequent genetic analysis of amplified fragments, information on microbiological systematics and phylogeny can be obtained in a fast and efficient manner. Similar types of gene identification can be used to verify or detect genes responsible for phenotypic characteristics, whereas modified forms of the PCR enable whole genome searches for genetic polymorphisms among strains of a given species. In medical sciences, both strategies, gene and genome variability analysis by PCR, have an increasing impact on the study of the spread of especially those microbes that are multiply resistant to clinically used antibiotics. In this communication we will exemplify the usefulness of PCR-mediated typing of microorganisms from a clinical perspective while focusing on gene- versus genome-scanning. Special emphasis will be placed on analysis of the dissemination and characteristics of methicillin-resistant Staphylococcus aureus (MRSA) strains and bacterial factors providing resistance to penicillin and other beta-lactam antibiotics. Technical limitations and possibilities for improvement will be discussed.     

On the consistency of a physical mapping method to reconstruct a chromosome in vitro

During recent years considerable effort has been invested in creating physical maps for a variety of organisms as part of the Human Genome Project and in creating various methods for physical mapping. The statistical consistency of a physical mapping method to reconstruct a chromosome, however, has not been investigated. In this paper, we first establish that a model of physical mapping by binary fingerprinting of DNA fragments is identifiable using the key assumption-for a large randomly generated recombinant DNA library, there exists a staircase of DNA fragments across the chromosomal region of interest. Then we briefly introduce epi-convergence theory of variational analysis and transform the physical mapping problem into a constrained stochastic optimization problem. By doing so, we prove epi-convergence of the physical mapping model and epi-convergence of the physical mapping method. Combining the identifiability of our physical mapping model and the epi-convergence of a physical mapping method, finally we establish strong consistency of a physical mapping method.     

Developmentally regulated gene expression of thrombomodulin in postimplantation mouse embryos

Embryonic lethality of thrombomodulin-deficient mice has indicated an essential role for this regulator of blood coagulation in murine development. Here, the embryonic expression pattern of thrombomodulin was defined by surveying beta-galactosidase activity in a mouse strain in which the reporter gene was placed under the regulatory control of the endogenous thrombomodulin promoter via homologous recombination in embryonic stem cells. The murine trophoblast was identified as a previously unrecognized anatomical site where TM expression is conserved between humans and mice and may exert a critical function during postimplantation development. Targeted reporter gene expression in mesodermal precursors of the endothelial cell lineage defined thrombomodulin as an early marker of vascular differentiation. Analysis of the thrombomodulin promoter in differentiating ES cells and in transgenic mice provided evidence for a disparate and cell type-specific gene regulatory control mechanism in the parietal yolk sac. The thrombomodulin promoter as defined in this study will allow the targeting of gene expression to the parietal yolk sac of transgenic mice and the initiation of investigations into the role of parietal endoderm in placental function.