Mouse Genome Projects Research Articles

Upstream – news in genomics

Since our last issue, several important genomes have been completely or ‘almost completely’ sequenced. The debate over the number of human genes has flared up once more, with one computational and one experimental study into the annotation of the human genome. The mouse genome project has a clone fingerprint map to aid their sequencing effort. The SAGE technique has been applied to Drosophila and the US National Science Foundation announced increased spending on plant genome research.

In situ hybridization for metalloproteinases and their inhibitors.

The imminent completion of the human and mouse genome projects will reveal all metalloproteases and metalloprotease inhibitors such as those belonging to the MMP, TIMP, ADAM, and ADAMTS () families. At the present time, there are over 65 published genes in these families and it is very likely that their total number will be much higher. Considerable effort will subsequently be required to undertake a systematic examination of the expression, function, and regulation of these genes.

Toward Chromosome-Level Comparative Analysis of Vertebrate Genomes

Since the publication of the human draft genome sequence, steady progress around the world is made to analyze the genome. In addition, mouse genome project and other vertebrate genome projects of model organisms are rapidly producing much genomic sequences. Comparative analysis is becoming increasingly important as a tool for genome sequence analysis [3]. In this study, we focus on vertebrate genome sequences of various species, such as human, mouse and rat. The evolutionary conserved regions in human genome sequences are identified effectively by the comparison with those vertebrate genome sequences. The main goal of this research is to identify novel genes and regulatory regions and to elucidate genome-level events in evolution from genome sequences. We have developed a comparative genome system which collects data, compares them, visualizes the results, and produces primer sets using primer3 [6] for the PCR experiments for confirmation of the exon prediction. Users can submit syntenic genomic sequences or select a publicly available syntenic region. The system allow the users to analyze and convert data easily, so that users can browse the various analysis results using PipMaker [4] (WWW server for genome comparison and java application laj), and Alfresco [2] (java application), without producing annotation files on their own.

Evaluating mutant mice: anatomic pathology.

As the human and mouse genome projects approach their goals, initiatives in functional genomics are advancing. When the nucleotide sequences are available, identification of gene functions will assume even greater importance. Determination of gene products and their proximal biochemical functions provide a part of the picture, but determination of their functions in the context of the whole organism is the ultimate goal. The manipulated mouse genome has become accepted as a model for understanding the genetic basis of human conditions and diseases. Consequently, biomedical research institutions have seen significant increases in the use of mice since the early 1980s, and these increases are largely attributable to the use of genetically modified mice. The role of comparative pathology in research on mutant mouse models of disease is increasing in response to these trends. Evaluation and phenotypic characterization of mutant mice, via clinical and anatomic pathology techniques, will be an important component of functional genomics initiatives.

Inherited Muscle Diseases Towards Improvements of the Mouse Model Catalogue

The muscle disease field has benefited enormously from the increasing availability of genomic resources derived from the human genome mapping and allied projects. Although a number of skeletal and cardiac disorders are still at the linkage or candidate gene analysis stage, genes have been identified for the most common ones. The increasing variety of genes involved makes genetic screens an essential tool for more precise diagnosis and better classification of these disorders. The mouse represents the system of choice for the study of muscular dystrophies and cardiomyopathies but, not surprisingly, many murine models often show phenotypic differences between human and mouse. Only a handful of spontaneous or targeted mouse mutants are available within the field of muscle diseases compared to the increasing genetic complexity of myopathies and cardiomyopathies identified in humans. This shortage has been identified in other inherited diseases and has contributed to the revival of ENU (ethylnitrosourea) mutagenesis. Several large-scale ENU mutagenesis programs set up worldwide should provide allelic collections of mutants for pathologically interesting genes and uncover new gene functions. An effective integration between high throughput production of mutants, DNA-chip based expression analysis and the comprehensive maps derived from the human and mouse genome projects will make possible systematic studies of mammalian gene function, facilitating the identification of better mouse models. Keywords: Muscle Diseases, Dystrophin glycoprotein complex DGC, Limb girdle Muscular dystrophy LGMD, Muscular dystrophy CMD, Cardiomyopathies, Mutagenesis, Muscle lim protein MLP

Establishment and chimera analysis of 129/SvEv- and C57BL/6-derived mouse embryonic stem cell lines.

Hundreds of new mutant mouse lines are being produced annually using gene targeting and gene trap approaches in embryonic stem (ES) cells, and the number is expected to continue to grow as the human and mouse genome projects progress. The availability of robust ES cell lines and a simple technology for making chimeras is more attractive now than ever before. We established several new ES cell lines from 129/SvEv and C57BL/6 mice and tested their ability to contribute to the germline following blastocyst injections and/or the less expensive and easier method of morula-ES cell aggregation. Using morula aggregation to produce chimeras, five newly derived 129/SvEv and two C57BL/6 ES cell lines tested at early passages were found to contribute extensively to chimeras and produce germline-transmitting male chimeras. Furthermore, the two 129S/vEv ES cell lines that were tested and one of the C57BL/6 ES cell lines were able to maintain these characteristics after many passages in vitro. Our results indicate that the ability of ES cells to contribute strongly to chimeras following aggregation with outbred embryos is a general property of early passage ES cells and can be maintained for many passages. C56BL/6-derived ES cell lines, however, have a greater tendency than 129-derived ES cell lines to lose their ability to colonize the germline.

Neurons from stem cells: Implications for understanding nervous system development and repair

Neurodegenerative diseases cost the economies of the developed world billions of dollars per annum. Given ageing population profiles and the increasing extent of this problem, there has been a surge of interest in neural stem cells and in neural differentiation protocols that yield neural cells for therapeutic transplantation. Due to the oncogenic potential of stem cells a better characterisation of neural differentiation, including the identification of new neurotrophic factors, is required. Stem cell cultures undergoing synchronous in vitro neural differentiation provide a valuable resource for gene discovery. Novel tools such as microarrays promise to yield information regarding gene expression in stem cells. With the completion of the yeast, C. elegans, Drosophila, human, and mouse genome projects, the functional characterisation of genes using genetic and bioinformatic tools will aid in the identification of important regulators of neural differentiation.

Genotype-based screen for ENU-induced mutations in mouse embryonic stem cells.

The ability to generate mutations is a prerequisite to functional genetic analysis. Despite a long history of using mice as a model system for genetic analysis, the scientific community has not generated a comprehensive collection of multiple alleles for most mouse genes. The chemical mutagen of choice for mouse has been N-ethyl-N-nitrosourea (ENU), an alkylating agent that mainly causes base substitutions in DNA, and therefore allows for recovery of complete and partial loss-, as well as gain-, of-function alleles . Specific locus tests designed to detect recessive mutations showed that ENU is the most efficient mutagen in mouse with an approximate mutation rate of 1 in 1,000 gametes. In fact, several genome-wide and region-specific screens based on phenotypes have been carried out. The anticipation of the completion of the human and mouse genome projects, however, now emphasizes genotype-driven genetics--from sequence to mutants. To take advantage of the mutagenicity of ENU and its ability to create allelic series of mutations, we have developed a complementary approach to generating mutations using mouse embryonic stem (ES) cells. We show that a high mutation frequency can be achieved and that modulating DNA-repair activities can enhance this frequency. The treated cells retain germline competency, thereby rendering this approach applicable for efficient generation of an allelic series of mutations pivotal to a fine-tuned dissection of biological pathways.

Finding the genes that direct mammalian development: ENU mutagenesis in the mouse

The genetic control of mammalian embryogenesis is not well understood. N-ethyl-N-nitrosourea (ENU) mutagenesis screens in the mouse provide a route to identify more of the genes that are required for mammalian development. The characterization of ENU-induced mutations can build on the resources provided by the mouse and human genome projects to help define the tissue interactions and signaling pathways that direct early mammalian development.

Neurons from stem cells: Implications for understanding nervous system development and repair

Neurodegenerative diseases cost the economies of the developed world billions of dollars per annum. Given ageing population profiles and the increasing extent of this problem, there has been a surge of interest in neural stem cells and in neural differentiation protocols that yield neural cells for therapeutic transplantation. Due to the oncogenic potential of stem cells a better characterisation of neural differentiation, including the identification of new neurotrophic factors, is required. Stem cell cultures undergoing synchronous in vitro neural differentiation provide a valuable resource for gene discovery. Novel tools such as microarrays promise to yield information regarding gene expression in stem cells. With the completion of the yeast, C. elegans, Drosophila, human, and mouse genome projects, the functional characterisation of genes using genetic and bioinformatic tools will aid in the identification of important regulators of neural differentiation.

Leukaemia disease genes: large-scale cloning and pathway predictions.

Retroviral insertional mutagenesis in BXH2 and AKXD recombinant inbred mice induces a high incidence of myeloid or B- and T-cell leukaemia and the proviral integration sites in the leukaemias provide powerful genetic tags for disease gene identification. Some of the disease genes identified by proviral tagging are also associated with human disease, validating this approach for human disease gene identification. Although many leukaemia disease genes have been identified over the years, many more remain to be cloned. Here we describe an inverse PCR (IPCR) method for proviral tagging that makes use of automated DNA sequencing and the genetic tools provided by the Mouse Genome Project, which increases the throughput for disease gene identification. We also use this IPCR method to clone and analyse more than 400 proviral integration sites from AKXD and BXH2 leukaemias and, in the process, identify more than 90 candidate disease genes. Some of these genes function in pathways already implicated in leukaemia, whereas others are likely to define new disease pathways. Our studies underscore the power of the mouse as a tool for gene discovery and functional genomics.

Linkage mapping of microdissected clones from distal mouse chromosome 16.

A total of 38 unique segments generated by microdissection of mouse chromosome 16 (MMU16), sequence independent amplification (SIA) and cloning were sequentially mapped on the distal portion of the chromosome with two mouse backcross panels. Some reference markers from other sources were retyped in the panels and results integrated with those for our microdissected DNA segments. The clone map is most highly refined in its distal portion, which stretches from reference marker D16Mit71 to D16Mit5, and the highest density of clones is in the region defined by markers D16Mit5 and D16Mit141. This map on distal mouse chromosome 16 should be a useful tool for the mouse genome project and for studies of genes in the region.

Cloning and expression of mouse 60 kDa ribonucleoprotein SS-A/Ro.

Autoantibodies to SS-A/Ro are among the most common found in sera of patients with systemic rheumatic diseases. These autoimmune diseases can affect various organ systems of the body and are variable in their manifestations and presentation. One of the autoimmune targets is the 60 kDa SS-A/Ro protein known to be associated with small cytoplasmic Y RNAs. To study systematically the expression of the protein, we have cloned the mouse full length 60 kDa SS-A/Ro cDNA using 5' RACE based on a cDNA sequence reported in the mouse genome project. The recombinant protein derived from the putative full-length construct was shown to react with human prototype anti-SS-A/Ro serum Ge in western blot and immunoprecipitation and comigrated with cellular 60 kDa SS-A/Ro protein in 3T3 cells. Cellular expression, measured by RT-PCR, was highest in mouse brain, followed by lung, muscle, kidney and heart. Lower levels were found in testis, liver and spleen. Like the human 60 kDa SS-A/Ro protein, the deduced mouse homolog has 538 amino acids. Sequence analysis showed 89.9% identity and 95.0% similarity between the mouse and human proteins.

Building an integrated mouse genome database: a view from the front line

The mouse is important as a model for human genetic processes and diseases due to the high degree of similarity between the two genomes. Here, the authors review their experiences and attempt to relay some of the lessons learned, technical and otherwise, in their efforts to build the Mouse Genome Database (MGD). The topics discussed include: the Mouse Genome Project; developing the MGD; online databases; Macintosh computer databases; flat file databases; the encyclopedia of the mouse genome; database types. >

Toward a whole cDNA catalog: construction of an equalized cDNA library from mouse embryos.

The construction of a cDNA library containing all genes without redundancy is one of the major technical challenges for biology. Toward this goal, we have developed an equalized (normalized) cDNA library from mRNA pools derived from mouse embryos that cover the entire period of mouse ontogenesis. Colony hybridization analyses with 11 genes showed the reduction of abundance variation from at least 6000-fold in the unequalized S-library to approximately 33-fold in the EIII-library, which was constructed after three cycles of equalization procedure. Limiting dilution PCR analyses with 26 tissue-specific genes showed the reduction of abundance variation from at least approximately 1,000,000-fold in the S-cDNA mixture to approximately 100-fold in the EIII-cDNA mixture. Based on these analyses, we estimate that at least 15,000 independent cDNA clones are included with little redundancy in the EIII-cDNA library. This will be a useful resource for mouse biology as well as the mouse genome project.

A plan for the mouse genome project

A plan for the mouse genome project

The mouse genome project and human genetics: A report from the 5th International Mouse Genome Mapping Workshop, Lunteren, Holland

• • Genome-wide mapping efforts are moving toward the establishment of a 1-cM genetic map of the entire mouse genome. • • The bulk of linkage groups conserved between the mouse and the human genomes has been identified. • • Microsatellite mapping has had a major impact on the development of genome-wide genetic maps and, in particular, on genome-wide searches for polygenic disease loci. • • Some substantial regions of the mouse genome have a marker density of 1 cM or less and many of these regions are now physically mapped. • • Embryonic YAC contigs have been established in some physically mapped regions. • • A unitary, global mouse mapping database—the Mouse Genome Database—is under development along with associated software tools. • • Chromosome committees are having a major impact on the establishment and verification of chromosome maps through the preparation of published annual reports.

Isolation of a large number of novel mammalian genes by a differential cDNA library screening strategy.

As part of the ongoing human and mouse genome projects, the aim of this study was to isolate novel, previously uncharacterized, genes from mouse testis. Two approaches were compared for their effectiveness in isolating novel genes: random, vs differential, complementary DNA (cDNA) cloning methods. In the differential approach, only the cDNA clones containing rare sequences (as determined by preliminary clone hybridization) are further analyzed; in the random approach, cDNA clones are isolated at random from the cDNA library. More than two hundred cDNA clones altogether were analyzed, using a PCR-mediated amplification and sequencing strategy. A comparison of these sequences to nucleic acid and protein sequence databases, revealed that 84% of the isolated rare cDNA clones represented new, previously uncharacterized mouse genes. In contrast, less than 63% of the cDNA clones isolated at random from cDNA libraries, contained novel genes. Thus, the probability of isolating new, previously uncharacterized, mammalian genes from cDNA libraries can be markedly improved by focusing efforts on clones containing rare sequences.