Checks and balancers: balancer chromosomes to facilitate genome annotation

Phenotype-driven mutagenesis screens are used to discover gene function in model organisms. Mutations that are induced by chemical mutagens can occur anywhere in the genome. However, the use of a balancer chromosome (where a phenotypically marked segment of a chromosome is inverted) in a mutagenesis screen enables mutations to be mapped in a defined region of the genome and maintained stably in a heterozygous state. Mouse balancer chromosomes can be engineered using Cre-loxP technology in selected regions of the genome. Balancer mutagenesis screens will provide a systematic functional analysis of the genes on mouse chromosomes, and consequently, will facilitate a functional annotation of the mammalian genome sequence.     

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     

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.     

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     

The expanding role of mouse genetics for understanding human biology and disease

It has taken about 100 years since the mouse first captured our imagination as an intriguing animal for it to become the premier genetic model organism. An expanding repertoire of genetic technology, together with sequencing of the genome and biological conservation, place the mouse at the foremost position as a model to decipher mechanisms underlying biological and disease processes. The combined approaches of embryonic stem cell-based technologies, chemical and insertional mutagenesis have enabled the systematic interrogation of the mouse genome with the aim of creating, for the first time, a library of mutants in which every gene is disrupted. The hope is that phenotyping the mutants will reveal novel and interesting phenotypes that correlate with genes, to define the first functional map of a mammalian genome. This new milestone will have a great impact on our understanding of mammalian biology, and could significantly change the future of medical diagnosis and therapeutic development, where databases can be queried in silico for potential drug targets or underlying genetic causes of illnesses. Emerging innovative genetic strategies, such as somatic genetics, modifier screens and humanized mice, in combination with whole-genome mutagenesis will dramatically broaden the utility of the mouse. More significantly, allowing genome-wide genetic interrogations in the laboratory, will liberate the creativity of individual investigators and transform the mouse as a model for making original discoveries and establishing novel paradigms for understanding human biology and disease.     

Mutagenesis strategies for identifying novel loci associated with disease phenotypes

The systematic identification of the function of all the genes in the mammalian genome is one of the major scientific challenges for the 21st century. A comprehensive insight into mammalian gene function will illuminate our understanding of the genetic bases of disease. Mouse mutagenesis is a powerful tool for the study of mammalian gene function. Most recently, a number of approaches employing the chemical mutagen ethylnitrosourea (ENU) have been utilised by mouse geneticists to deliver a substantial new collection of mouse disease models. The growing mouse mutant archive provides a powerful resource for the identification of novel genes involved with human genetic disease.     

Mice and humans: chromosome engineering and its application to functional genomics

Functional modeling of human genes and diseases requires suitable mammalian model organisms. For its genetic malleability, the mouse is likely to continue to play a major role in defining basic genetic traits and complex pathological disorders. Recently, gene targeting techniques have been extended towards developing new engineering strategies for generating extensive lesions and rearrangements in mouse chromosomes. While these advances create new opportunities to address similar aberrations observed in human diseases, they also open new ways of scaling-up mutagenesis projects that try to catalogue and annotate cellular functions of mammalian genes.     

Conditional gene targeting in the kidney

Complete mapping of the genome in a number of organisms provides a challenge for experimental nephrologists to identify potential functions of a vast number of new genes in the kidney. Since knockout technologies have evolved in the early eighties the mouse has become a valuable model organism. Researchers can now artificially eliminate the expression of specific genes in a mammalian organism and examine the phenotype. New developments have emerged that allow investigators to knock out a gene specifically in the kidney. Several kidney-specific promoters provide valuable tools and bacterial artificial chromosome (BAC) based techniques like recombineering will enhance both number and accuracy of new mouse lines with spatially controlled gene expression. In addition to spatial control, tetracycline- or tamoxifen-inducible systems, provide the possibility of influencing the temporal expression pattern of a gene enabling researchers to dissect its functions in adult organisms. Knocking out a gene will continue to be the gold standard for defining the role of a specific gene whereas tissue-specific gene knockdown using RNA interference represents an alternative approach for generating lower-priced and fast loss of function models. In addition to reverse genetic approaches, forward genetic techniques like random mutagenesis in mice continue to evolve and will enhance our understanding of disease mechanisms in the kidney.     

The Mouse Genome Database (MGD): integration nexus for the laboratory mouse

The Mouse Genome Database (MGD) is the community database resource for the laboratory mouse, a key model organism for interpreting the human genome and for understanding human biology and disease (http://www.informatics.jax.org). MGD provides standard nomenclature and consensus map positions for mouse genes and genetic markers; it provides a curated set of mammalian homology records, user-defined chromosomal maps, experimental data sets and the definitive mouse 'gene to sequence' reference set for the research community. The integration and standardization of these data sets facilitates the transition between mouse DNA sequence, gene and phenotype annotations. A recent focus on allele and phenotype representations enhances the ability of MGD to organize and present data for exploring the relationship between genotype and phenotype. This link between the genome and the biology of the mouse is especially important as phenotype information grows from large mutagenesis projects and genotype information grows from large-scale sequencing projects.     

Comparative genetic analysis: the utility of mouse genetic systems for studying human monogenic disease

One of the long-term goals of mutagenesis programs in the mouse has been to generate mutant lines to facilitate the functional study of every mammalian gene. With a combination of complementary genetic approaches and advances in technology, this aim is slowly becoming a reality. One of the most important features of this strategy is the ability to identify and compare a number of mutations in the same gene, an allelic series. With the advent of gene-driven screening of mutant archives, the search for a specific series of interest is now a practical option. This review focuses on the analysis of multiple mutations from chemical mutagenesis projects in a wide variety of genes and the valuable functional information that has been obtained from these studies. Although gene knockouts and transgenics will continue to be an important resource to ascertain gene function, with a significant proportion of human diseases caused by point mutations, identifying an allelic series is becoming an equally efficient route to generating clinically relevant and functionally important mouse models.     

Insertional mutagenesis in mice: new perspectives and tools

Insertional mutagenesis has been at the core of functional genomics in many species. In the mouse, improved vectors and methodologies allow easier genome-wide and phenotype-driven insertional mutagenesis screens. The ability to generate homozygous diploid mutations in mouse embryonic stem cells allows prescreening for specific null phenotypes prior to in vivo analysis. In addition, the discovery of active transposable elements in vertebrates, and their development as genetic tools, has led to in vivo forward insertional mutagenesis screens in the mouse. These new technologies will greatly contribute to the speed and ease with which we achieve complete functional annotation of the mouse genome.     

Identification and analysis of genes and pseudogenes within duplicated regions in the human and mouse genomes

The identification and classification of genes and pseudogenes in duplicated regions still constitutes a challenge for standard automated genome annotation procedures. Using an integrated homology and orthology analysis independent of current gene annotation, we have identified 9,484 and 9,017 gene duplicates in human and mouse, respectively. On the basis of the integrity of their coding regions, we have classified them into functional and inactive duplicates, allowing us to define the first consistent and comprehensive collection of 1,811 human and 1,581 mouse unprocessed pseudogenes. Furthermore, of the total of 14,172 human and mouse duplicates predicted to be functional genes, as many as 420 are not included in current reference gene databases and therefore correspond to likely novel mammalian genes. Some of these correspond to partial duplicates with less than half of the length of the original source genes, yet they are conserved and syntenic among different mammalian lineages. The genes and unprocessed pseudogenes obtained here will enable further studies on the mechanisms involved in gene duplication as well as of the fate of duplicated genes.     

Efficient Generation of Germ Line Transmitting Chimeras from C57BL/6N ES Cells by Aggregation with Outbred Host Embryos

Genetically modified mouse strains derived from embryonic stem (ES) cells have become essential tools for functional genomics and biomedical research. Large scale mutagenesis projects are producing libraries of mutant C57BL/6 (B6) ES cells to enable the functional annotation of every gene of the mouse genome. To realize the utility of these resources, efficient and accessible methods of generating mutant mice from these ES cells are necessary. Here, we describe a combination of ICR morula aggregation and a chemically-defined culture medium with widely available and accessible components for the high efficiency generation of germline transmitting chimeras from C57BL/6N ES cells. Together these methods will ease the access of the broader biomedical research community to the publicly available B6 ES cell resources.     

Mouse splice mutant generation from ENU-treated ES cells--a gene-driven approach

Mutant mice are important for elucidating mammalian gene functions and for modeling human disease phenotypes. In recent years, chemical mutagenesis has become an increasingly popular method to disrupt gene functions due to its high efficiency of inducing mutations throughout the genome. Mutagenesis of embryonic stem (ES) cells offers the possibility of gene-driven approaches, which, however, require efficient mutation detection procedures to screen archives of mutated samples for lesions in particular genes. We have developed an approach that focuses on the detection of splice mutations in highly pooled cDNA samples using exon-skipping PCR primers. As a proof of concept, splice mutants for the Kit gene were isolated from a library comprising approximately 40,000 ES cell clones treated with N-ethyl-N-nitrosourea followed by transmission through the mouse germ-line. The approach will be useful for the production of mouse models for human disease-related splice mutations and as a general gene disruption strategy.     

MaGe: a microbial genome annotation system supported by synteny results

Magnifying Genomes (MaGe) is a microbial genome annotation system based on a relational database containing information on bacterial genomes, as well as a web interface to achieve genome annotation projects. Our system allows one to initiate the annotation of a genome at the early stage of the finishing phase. MaGe's main features are (i) integration of annotation data from bacterial genomes enhanced by a gene coding re-annotation process using accurate gene models, (ii) integration of results obtained with a wide range of bioinformatics methods, among which exploration of gene context by searching for conserved synteny and reconstruction of metabolic pathways, (iii) an advanced web interface allowing multiple users to refine the automatic assignment of gene product functions. MaGe is also linked to numerous well-known biological databases and systems. Our system has been thoroughly tested during the annotation of complete bacterial genomes (Acinetobacter baylyi ADP1, Pseudoalteromonas haloplanktis, Frankia alni) and is currently used in the context of several new microbial genome annotation projects. In addition, MaGe allows for annotation curation and exploration of already published genomes from various genera (e.g. Yersinia, Bacillus and Neisseria). MaGe can be accessed at http://www.genoscope.cns.fr/agc/mage.     

Generation of Radiation-Induced Deletion Complexes in the Mouse Genome Using Embryonic Stem Cells

As the genetic and physical mapping stage of the Human Genome Project nears completion, the focus is shifting toward the development of technologies for high-throughput analysis of gene function. Whereas DNA sequencing will enable the assignment of presumed function to a large number of genes in mice and humans, it is clear that the great majority of genes will have to be evaluatedin vivoto accurately assess their role in a complex organism. While gene targeting in mouse embryonic stem (ES) cells is the current method of choice for the characterization of gene function in mice, it remains relatively labor intensive and lacks the throughput required for analysis of genome function on a large scale. Alternative methods of efficient mutagenesis will clearly be required for this task. Chromosomal deletions are powerful tools in the genetic analysis of complex genomes, enabling the systematic identification and localization of functional units along defined chromosomal regions. Not only are deletions useful for the identification of genetic functions, but they serve as mapping reagents for existing mutations or traits. While their use has been an essential tool inDrosophilagenetics, classical mutagenesis in mice has been logistically impractical for generating deletions. We have previously described an efficient method for generating radiation-induced deletion complexes at defined regions in the genome using ES cells. In this article, we detail the methodological aspects of this technology and describe the applications of chromosomal deletions for characterizing gene function in ways that make optimal use of the information generated by the first stage of the Genome Project.     

Overlapping deletions spanning the proximal two-thirds of the mouse t complex

Chromosome deletion complexes in model organisms serve as valuable genetic tools for the functional and physical annotation of complex genomes. Among their many roles, deletions can serve as mapping tools for simple or quantitative trait loci (QTLs), genetic reagents for regional mutagenesis experiments, and, in the case of mice, models of human contiguous gene deletion syndromes. Deletions also are uniquely suited for identifying regions of the genome containing haploinsufficient or imprinted loci. Here we describe the creation of new deletions at the proximal end of mouse Chromosome (Chr) 17 by using the technique of ES cell irradiation and the extensive molecular characterization of these and previously isolated deletions that, in total, cover much of the mouse t complex. The deletions are arranged in five overlapping complexes that collectively span about 25 Mbp. Furthermore, we have integrated each of the deletion complexes with physical data from public and private mouse genome sequences, and our own genetic data, to resolve some discrepancies. These deletions will be useful for characterizing several phenomena related to the t complex and t haplotypes, including transmission ratio distortion, male infertility, and the collection of t haplotype embryonic lethal mutations. The deletions will also be useful for mapping other loci of interest on proximal Chr 17, including T-associated sex reversal ( Tas) and head-tilt ( het). The new deletions have thus far been used to localize the recently identified t haplolethal ( Thl1) locus to an approximately 1.3-Mbp interval.     

Comparative genomics approach to detecting split-coding regions in a low-coverage genome: lessons from the chimaera Callorhinchus milii (Holocephali, Chondrichthyes)

Recent development of deep sequencing technologies has facilitated de novo genome sequencing projects, now conducted even by individual laboratories. However, this will yield more and more genome sequences that are not well assembled, and will hinder thorough annotation when no closely related reference genome is available. One of the challenging issues is the identification of protein-coding sequences split into multiple unassembled genomic segments, which can confound orthology assignment and various laboratory experiments requiring the identification of individual genes. In this study, using the genome of a cartilaginous fish, Callorhinchus milii, as test case, we performed gene prediction using a model specifically trained for this genome. We implemented an algorithm, designated ESPRIT, to identify possible linkages between multiple protein-coding portions derived from a single genomic locus split into multiple unassembled genomic segments. We developed a validation framework based on an artificially fragmented human genome, improvements between early and recent mouse genome assemblies, comparison with experimentally validated sequences from GenBank, and phylogenetic analyses. Our strategy provided insights into practical solutions for efficient annotation of only partially sequenced (low-coverage) genomes. To our knowledge, our study is the first formulation of a method to link unassembled genomic segments based on proteomes of relatively distantly related species as references.