Molecular genetics of the Pax gene family

Three members of the Pax gene family are now known to be responsible for the established mouse developmental phenotypes Splotch, Small eye and undulated; two of these genes are implicated in the human congenital diseases Waardenburg's syndrome and aniridia. The mouse mutants will act as model systems for these human disorders and, in addition, will provide insights into the processes of vertebrate development.     

Functional and evolutionary analyses on expressed intronless genes in the mouse genome

Using computational approaches we have identified 2017 expressed intronless genes in the mouse genome. Evolutionary analysis reveals that 56 intronless genes are conserved among the three domains of life--bacteria, archea and eukaryotes. These highly conserved intronless genes were found to be involved in essential housekeeping functions. About 80% of expressed mouse intronless genes have orthologs in eukaryotic genomes only, and thus are specific to eukaryotic organisms. 608 of these genes have intronless human orthologs and 302 of these orthologs have a match in OMIM database. Investigation into these mouse genes will be important in generating mouse models for understanding human diseases.     

Genetic Models in Applied Physiology. Functional genomics in the mouse: powerful techniques for unraveling the basis of human development and disease.

Now that near-complete DNA sequences of both the mouse and human genomes are available, the next major challenge will be to determine how each of these genes functions, both alone and in combination with other genes in the genome. The mouse has a long and rich history in biological research, and many consider it a model organism for the study of human development and disease. Over the past few years, exciting progress has been made in developing techniques for chromosome engineering, mutagenesis, mapping and maintenance of mutations, and identification of mutant genes in the mouse. In this mini-review, many of these powerful techniques will be presented along with their application to the study of development, physiology, and disease.     

The right time,the right place: will targeting human cancer-associated mutations to the mouse provide the perfect preclinical model?

Over the past 10 years the realisation that genetic mouse models of cancer may play a key role in preclinical drug development has gained strong momentum. Moreover sequencing studies of human tumours have provided key insights into the mutational complexity of epithelial cancer, unleashing important clues for researchers to generate accurate genetically engineered mouse (GEM) models of cancer. Thus by targeting multiple cancer associated human mutations to the appropriate murine epithelia, mice develop tumours that more closely recapitulate the human disease. As a number of excellent models now exist, the next 5-10 years will ascertain whether these models will predict response of human cancer to intervention. If so they might become the 'gold standard' where all drugs are required to be tested in mouse models of disease before proceeding into the patient. However, although this principle is very attractive, it is relatively untested and here, using examples of prevalent human cancers, we will review the latest data on preclinical GEM studies and comment on what challenges are left to overcome.     

Identifying novel genes for atherosclerosis through mouse-human comparative genetics

Susceptibility to atherosclerosis is determined by both environmental and genetic factors. Its genetic determinants have been studied by use of quantitative-trait-locus (QTL) analysis. So far, 21 atherosclerosis QTLs have been identified in the mouse: 7 in a high-fat-diet model only, 9 in a sensitized model (apolipoprotein E- or LDL [low-density lipoprotein] receptor-deficient mice) only, and 5 in both models, suggesting that different gene sets operate in each model and that a subset operates in both. Among the 27 human atherosclerosis QTLs reported, 17 (63%) are located in regions homologous (concordant) to mouse QTLs, suggesting that these mouse and human atherosclerosis QTLs have the same underlying genes. Therefore, genes regulating human atherosclerosis will be found most efficiently by first finding their orthologs in concordant mouse QTLs. Novel mouse QTL genes will be found most efficiently by using a combination of the following strategies: identifying QTLs in new crosses performed with previously unused parental strains; inducing mutations in large-scale, high-throughput mutagenesis screens; and using new genomic and bioinformatics tools. Once QTL genes are identified in mice, they can be tested in human association studies for their relevance in human atherosclerotic disease.     

Trans-NIH neuroscience initiatives on mouse phenotyping and mutagenesis

In the post-genomic era, the laboratory mouse will excel as a premier mammalian system to study normal and disordered biological processes, in part because of low cost, but largely because of the rich opportunities that exist for exploiting genetic tools and technologies in the mouse to systematically determine mammalian gene function. Many robust models of human disease may therefore be developed, and these in turn will provide critical clues to understanding gene function. The full potential of the mouse for understanding many of the neural and behavioral phenotypes of relevance to neuroscientists has yet to be realized. With the full anatomy of the mouse genome at hand, researchers for the first time will be able to move beyond traditional gene-by-gene approaches and take a global view of gene expression patterns crucial for neurobiological processes. In response to an action plan for mouse genomics developed on the basis of recommendations from the scientific community, seven institutes of the National Institutes of Health (NIH) initiated in 1999 a mouse genetics research program that specifically focused on neurobiology and complex behavior. The specific goals of these neuroscience initiatives are to develop high-throughput phenotyping assays and to initiate genome-wide mutagenesis projects to identify hundreds of mutant strains with heritable abnormalities of high relevance to neuroscientists. Assays and mutants generated in these efforts will be made widely available to the scientific community, and such resources will provide neuroscientists unprecedented opportunities to elucidate the molecular mechanisms of neural function and complex behavior. Such research tools ultimately will permit the manipulation and analysis of the mouse genome, as a means of gaining insight into the genetic bases of the mammalian nervous system and its complex disorders. Received: 10 April 2001 / Accepted: 23 April 2001     

Species specificity of amidine-based urokinase inhibitors

Inhibition of the proteolytic activity of urokinase has been shown to inhibit the progression of tumors in rodent models and is being investigated for use in human disease. Understanding the rodent/human species-specificity of urokinase inhibitors is therefore critical for interpretation of rodent cancer progression models that use these inhibitors. We report here studies with a panel of 11 diverse urokinase inhibitors in both human and mouse enzymatic assays. Inhibitors such as amiloride, B428, and naphthamidine, that occupy only the S1 subsite pocket were found to be nearly equipotent between the human and the murine enzymes. Inhibitors that access additional, more distal, pockets were significantly more potent against the human enzyme but there was no corresponding potency increase against the murine enzyme. X-ray crystallographic structures of these compounds bound to the serine protease domain of human urokinase were solved and examined in order to explain the human/mouse potency differences. The differences in inhibitor potency could be attributed to four amino acid residues that differ between murine and human urokinases: 60, 99, 146, and 192. These residues are Asp, His, Ser, and Gln in human and Gln, Tyr, Glu, and Lys in mouse, respectively. Compounds bearing a cationic group that interacts with residue 60 will preferentially bind to the human enzyme because of favorable electrostatic interactions. The hydrogen bonding to residue 192 and steric considerations with residues 99 and 146 also contribute to the species specificity. The nonparallel human/mouse enzyme inhibition observations were extended to a cell-culture assay of urokinase-activated plasminogen-mediated fibronectin degradation with analogous results. These studies will aid the interpretation of in vivo evaluation of urokinase inhibitors.     

Use of genetically modified mouse models for evaluation of carcinogenic risk: considerations for the laboratory animal scientist

There has been increasing interest in the use of selected genetically modified (GM) mouse models for the testing of chemicals to determine their carcinogenic potential. GM mouse models are believed to be useful tools that offer mechanistically relevant insights for understanding and predicting the human response to chemical exposure. They have been proposed as alternatives to the traditional 2-year mouse oncogenicity bioassay. In this overview we will review the GM mouse models that have been proposed as bioassay alternatives and present some of the key laboratory animal science challenges that need to be considered when using these unique animals.     

Genomics meets genetics: towards a mutant map of the mouse

Phenotype-driven mutagenesis approaches in the mouse will deliver a vastly expanded mouse mutant resource and can be expected to lead to the identification of novel genes and pathways, enabling the emergence of new insights into mammalian gene function. In order for this goal to be realized, developments in genomics need to be harnessed to progress in mouse mutagenesis. We need firstly to generate a mutant map of the mouse, devising and employing rapid methods for the genetic mapping of the growing mouse mutant resource. Secondly, we need to be able to rapidly identify and assess candidate genes in the vicinity of the mapped mutations. Developments in mapping and genotyping technology are described that will potentially speed the construction of a rich mutant map of the mouse. In addition, the benefits of comparative sequencing of the human and mouse genomes are reviewed. The availability of both human and mouse genome sequences will underpin the evolution of a comprehensive and well annotated mammalian gene map that will significantly enhance our ability to move rapidly from mapped mutation to the identification of the underlying gene. Received: 16 December 1999 / Accepted: 17 December 1999     

Muscular dystrophies involving the dystrophin-glycoprotein complex: an overview of current mouse models

The dystrophin-glycoprotein complex (DGC) is a multisubunit complex that connects the cytoskeleton of a muscle fiber to its surrounding extracellular matrix. Mutations in the DGC disrupt the complex and lead to muscular dystrophy. There are a few naturally occurring animal models of DGC-associated muscular dystrophy (e.g. the dystrophin-deficient mdx mouse, dystrophic golden retriever dog, HFMD cat and the delta-sarcoglycan-deficient BIO 14.6 cardiomyopathic hamster) that share common genetic protein abnormalities similar to those of the human disease. However, the naturally occurring animal models only partially resemble human disease. In addition, no naturally occurring mouse models associated with loss of other DGC components are available. This has encouraged the generation of genetically engineered mouse models for DGC-linked muscular dystrophy. Not only have analyses of these mice led to a significant improvement in our understanding of the pathogenetic mechanisms for the development of muscular dystrophy, but they will also be immensely valuable tools for the development of novel therapeutic approaches for these incapacitating diseases.     

Large scale ENU screens in the mouse: genetics meets genomics

The worldwide effort to completely sequence the human and mouse genome will be accomplished within the next years. The focus of current activities within the framework of human genome research is mainly on the assembly of high resolution genetic and physical maps and genomic sequencing. Cloning of new genes is getting more easy using those maps. Nevertheless, it is necessary to work on a systematic analysis of gene function. Results obtained from these efforts will be of enormous value for future biological and biomedical research. However, even the complete sequence will not in all cases reveal the molecular and cellular role of the different genes. Therefore, the next phase of the Human Genome Project will have at its core the functional analysis of genes. Those genes relevant for the diagnosis, prevention and therapy of human diseases are of particular interest. Looking at the history of life sciences, mutants have been the most important tool to obtain insight into the biological function of genes. The mouse is the model of choice for the study of inherited diseases in man. In order to meet the requirements for functional human genome analysis, we need a large number of mouse mutants similar to the collection of mutants available for other model organisms such as flys and worms. To fully apply the power of genetics, multiple alleles of the same gene such as hypomorphs or hypermorphs are required. Efficient production of mouse mutants showing specific phenotypes can be achieved by the use of ethylnitrosourea (ENU). ENU is the most powerful mutagen known and we currently see a renaissance of ENU mutagenesis. The application of ENU mutagenesis is reviewed and discussed in the context of a new era of functional genomics.     

Human sperm do not bind to rat zonae pellucidae despite the presence of four homologous glycoproteins

The specificity of sperm-egg recognition in mammals is mediated primarily by the zona pellucida surrounding ovulated eggs. Mouse sperm are quite promiscuous and bind to human eggs, but human spermatozoa will not bind to mouse eggs. The mouse zona pellucida contains three glycoproteins, ZP1, ZP2, and ZP3, which are conserved in rat and human. The recent observation that human zonae pellucidae contain a fourth protein raises the possibility that the presence of four zona proteins will support human sperm binding. Using mass spectrometry, four proteins that are similar in size and share 62-70% amino acid identity with human ZP1, ZP2, ZP3, and ZP4/ZPB were detected in rat zonae pellucidae. However, although mouse and rat spermatozoa bind to eggs from each rodent, human sperm bind to neither, and the presence of human follicular fluid did not alter the specificity of sperm binding. In addition, mutant mouse eggs lacking hybrid/complex N-glycans or deficient in Core 2 O-glycans were no more able to support human sperm binding than normal mouse eggs. These data suggest that the presence of four zona proteins are not sufficient to support human sperm binding to rodent eggs and that additional determinants must be responsible for taxon-specific fertilization among mammals.     

Nucleotide excision repair- and p53-deficient mouse models in cancer research

Cancer is caused by the loss of controlled cell growth due to mutational (in)activation of critical genes known to be involved in cell cycle regulation. Three main mechanisms are known to be involved in the prevention of cells from becoming cancerous; DNA repair and cell cycle control, important to remove DNA damage before it will be fixed into mutations and apoptosis, resulting in the elimination of cells containing severe DNA damage. Several human syndromes are known to have (partially) deficiencies in these pathways, and are therefore highly cancer prone. Examples are xeroderma pigmentosum (XP) caused by an inborn defect in the nucleotide excision repair (NER) pathway and the Li-Fraumeni syndrome, which is the result of a germ line mutation in the p53 gene. XP patients develop skin cancer on sun exposed areas at a relatively early age, whereas Li-Fraumeni patients spontaneously develop a wide variety of early onset tumors, including sarcomas, leukemia's and mammary gland carcinomas. Several mouse models have been generated to mimic these human syndromes, providing us information about the role of these particular gene defects in the tumorigenesis process. In this review, spontaneous phenotypes of mice deficient for nucleotide excision repair and/or the p53 gene will be described, together with their responses upon exposure to either chemical carcinogens or radiation. Furthermore, possible applications of these and newly generated mouse models for cancer will be given.     

Genetic analysis of Down syndrome facilitated by mouse chromosome engineering

Human trisomy 21 is the most frequent live-born human aneuploidy and causes a constellation of disease phenotypes classified as Down syndrome, which include heart defects, myeloproliferative disorder, cognitive disabilities and Alzheimer-type neurodegeneration. Because these phenotypes are associated with an extra copy of a human chromosome, the genetic analysis of Down syndrome has been a major challenge. To complement human genetic approaches, mouse models have been generated and analyzed based on evolutionary conservation between the human and mouse genomes. These efforts have been greatly facilitated by Cre/loxP-mediated mouse chromosome engineering, which may result in the establishment of minimal critical genomic regions and eventually new dosage-sensitive genes associated with Down syndrome phenotypes. The success in genetic analysis of Down syndrome will further enhance our understanding of this disorder and lead to better strategies in developing effective therapeutic interventions.     

Technical approaches for mouse models of human disease

The mouse is the leading organism for disease research. A rich resource of genetic variation occurs naturally in inbred and special strains owing to spontaneous mutations. However, one can also obtain desired gene mutations by using the following processes: targeted mutations that eliminate function in the whole organism or in a specific tissue; forward genetic screens using chemicals or transposons; or the introduction of exogenous transgenes as DNAs, bacterial artificial chromosomes (BACs) or reporter constructs. The mouse is the only mammal that provides such a rich resource of genetic diversity coupled with the potential for extensive genome manipulation, and is therefore a powerful application for modeling human disease. This poster review outlines the major genome manipulations available in the mouse that are used to understand human disease: natural variation, reverse genetics, forward genetics, transgenics and transposons. Each of these applications will be essential for understanding the diversity that is being discovered within the human population.     

Derivation and characterization of pluripotent cell lines from pig embryos of different origins

Embryonic stem cells (ESCs) hold great promise for therapeutic use and represent a unique tool for investigating the process of self-renewal and differentiation. The properties that make ESCs unique are their capacity of unlimited self-renewal coupled with the property of re-entering the developmental process if returned inside a blastocyst. Such plasticity enable ESCs to form all embryonic tissues including germ cells. However, these remarkable properties, at present, have been demonstrated only for mouse ESCs even if cells with somehow more limited capacities have been derived in many different species including humans. The isolation of pluripotent embryonic cells lines from human embryos marked a crucial change of perspective in evaluating the properties defining an embryonic stem cell lines moving the focus from the generation of a germ-line chimera, obviously not feasible nor desirable in human, to the capacity of these cells to differentiate both in vivo and in vitro in fully mature and functional cell types of all kinds. Therefore, ESCs properties in species different from the mouse are being reassessed and re-evaluated, in view of their potential use as experimental models for the development of clinical applications. Among the species that may play a useful role in this field, the pig has a long-standing history as a prime animal model for pre-clinical biomedical applications and therefore, pig ESCs are attracting renewed interest. In this review, we will summarize the current knowledge on this topic and will contrast the relatively limited data available in this species with the much larger wealth of information available for mouse and human ESCs, in an attempt to assess whether or not pig ESCs can actually become a useful tool in the fast growing field of cell therapy.