Chemical mutagenesis and fine-structure functional analysis of the mouse genome

Heritable mutations constitute important raw materials for mammalian developmental genetics and general genome studies. Mutations induced by high-efficiency chemical mutagenesis of germ cells in mice can be used in genetic and molecular studies to complement physical-mapping strategies and to examine the nature and extent of the functional complexities hidden within the mammalian genome.     

Capitalizing on large-scale mouse mutagenesis screens

Variation is the crux of genetics. Mutagenesis screens in organisms from bacteria to fish have provided a battery of mutants that define protein functions within complex pathways. Large-scale mutation isolation has been carried out in Caenorhabditis elegans, Drosophila melanogaster and zebrafish, and has been recently reported in the mouse in two screens that have generated many new, clinically relevant mutations to reveal the power of phenotype-driven screens in a mammal.     

Chemical mutagenesis of the mouse genome: an overview

The careful comparison of the phenotypic variations generated by different alleles at a given locus, including of course, those alleles with a deleterious effect, is often an important source of information for the understanding of gene functions. In fact, every time it is possible to match a specific alteration observed at the genomic level with a particular pathology, it is possible to establish a relationship between a gene and its function. When considered from this point of view, the production of new mutations by experimental mutagenesis appears as an alternative to the strategy of in vitro gene invalidation by homologous recombination in embryonic stem (ES) cells, with the advantage that experimental mutagenesis does not require any previous knowledge of the gene structure at the molecular level. Homologous recombination in ES cells is a gene driven approach, in which mutant alleles are produced for those genes that we already know. Experimental mutagenesis, on the contrary, is a phenotype driven approach, in which unknown genes are identified based on phenotypic changes. Also, while homologous recombination in ES cells requires a rather sophisticated technology, mutagenesis is simple to achieve but relies greatly on the efficiency of the mutagenic treatment as well as on the use of an accurate protocol for phenotyping. In this review, we will address a few comments about the different techniques that can be used for the induction of point mutations in the mouse germ line with special emphasis on chemical mutagenesis. We will also discuss the limitations of experimental mutagenesis and the necessity to look for alternative ways for the discovery of new genes and gene functions in the mouse.     

Functional genomics in Arabidopsis: large-scale insertional mutagenesis complements the genome sequencing project

The ultimate goal of genome research on the model flowering plant Arabidopsis thaliana is the identification of all of the genes and understanding their functions. A major step towards this goal, the genome sequencing project, is nearing completion; however, functional studies of newly discovered genes have not yet kept up to this pace. Recent progress in large-scale insertional mutagenesis opens new possibilities for functional genomics in Arabidopsis. The number of T-DNA and transposon insertion lines from different laboratories will soon represent insertions into most Arabidopsis genes. Vast resources of gene knockouts are becoming available that can be subjected to different types of reverse genetics screens to deduce the functions of the sequenced genes.     

Implementation of a large-scale ENU mutagenesis program: towards increasing the mouse mutant resource

Systematic approaches to mouse mutagenesis will be vital for future studies of gene function. We have begun a major ENU mutagenesis program incorporating a large genome-wide screen for dominant mutations. Progeny of ENU-mutagenized mice are screened for visible defects at birth and weaning, and at 5 weeks of age by using a systematic and semi-quantitative screening protocol—SHIRPA. Following this, mice are screened for abnormal locomotor activity and for deficits in prepulse inhibition of the acoustic startle response. Moreover, in the primary screen, blood is collected from mice and subjected to a comprehensive clinical biochemical analysis. Subsequently, secondary and tertiary screens of increasing complexity can be used on animals demonstrating deficits in the primary screen. Frozen sperm is archived from all the male mice passing through the screen. In addition, tail tips are stored for DNA. Overall, the program will provide an extensive new resource of mutant and phenotype data to the mouse and human genetics communities at large. The challenge now is to employ the expanding mouse mutant resource to improve the mutant map of the mouse. An improved mutant map of the mouse will be an important asset in exploiting the growing gene map of the mouse and assisting with the identification of genes underlying novel mutations—with consequent benefits for the analysis of gene function and the identification of novel pathways. Received: 16 December 1999 / Accepted: 16 December 1999     

Trends in large-scale mouse mutagenesis: from genetics to functional genomics

The primary goal of mouse mutagenesis programmes is to develop a fundamental research infrastructure for mammalian functional genomics and to produce human disease models. Many large-scale programmes have been ongoing since 1997; these culminated in the International Knockout Mouse Consortium (IKMC) in 2007 with the aim to establish knockout and conditional mouse strains for all mouse genes. This article traces the origins and rationale of these large-scale mouse mutagenesis programmes.     

A two-component enhancer-inhibitor transposon mutagenesis system for functional analysis of the Arabidopsis genome.

A modified Enhancer-Inhibitor transposon system was used to generate a series of mutant lines by single-seed descent such that multiple I insertions occurred per plant. The distribution of original insertions in the population was assessed by isolating transposon-flanking DNA, and a database of insertion sites was created. Approximately three-quarters of the identified insertion sites show similarity to sequences stored in public databases, which demonstrates the power of this regimen of insertional mutagenesis. To isolate insertions in specific genes, we developed three-dimensional pooling and polymerase chain reaction strategies that we then validated by identifying mutants for the regulator genes APETALA1 and SHOOT MERISTEMLESS. The system then was used to identify inserts in a class of uncharacterized genes involved in lipid biosynthesis; one such insertion conferred a fiddlehead mutant phenotype.     

Random mutagenesis of the mouse genome: a strategy for discovering gene function and the molecular basis of disease

Mutagenesis of mice with N-ethyl-N-nitrosourea (ENU) is a phenotype-driven approach to unravel gene function and discover new biological pathways. Phenotype-driven approaches have the advantage of making no assumptions about the function of genes and their products and have been successfully applied to the discovery of novel gene-phenotype relationships in many physiological systems. ENU mutagenesis of mice is used in many large-scale and more focused projects to generate and identify novel mouse models for the study of gene functions and human disease. This review examines the strategies and tools used in ENU mutagenesis screens to efficiently generate and identify functional mutations.     

The paternal genome in mouse zygotes is less sensitive to ENU mutagenesis than the maternal genome

The two parental genomes lie separate within the zygote and may be differentially affected by environmental influences. We have shown earlier (Russell et al., 1988) that the maternal genome within the mouse zygote is exquisitely sensitive to the induction of point mutations by N-ethyl-N-nitrosourea (ENU), and that the initial lesion probably occurs in one strand of the DNA. The present experiment measured specific-locus mutation induction in the paternal genome. Zygotes containing a multiple-recessive maternal genome (a; b; p cch; d se; s) and the corresponding wild-type alleles in the paternal one were exposed to 50 mg ENU/kg in vivo at one of two stages: the presumed times of sperm entry and early pronuclear stage. At weaning age, the resulting mice were examined for mutations at the marked loci as well as for other mutations producing externally visible phenotypes. At the marked loci, one possible mosaic (for b) was observed among 2113 classified offspring that had been treated with ENU as zygotes; this animal failed to transmit a mutation. By contrast, in the reciprocal cross (which tests the maternal genome) we had observed 8 specific-locus mutations (6 of them mosaics) among 1555 offspring that had received the same dose of ENU during sperm entry (and completion of oocyte meiosis II). In the present experiment, we also found one mutation at other loci (two at other loci in the reciprocal cross). The frequency of offspring with small white belly spots was significantly greater in the treated groups (3.5 and 1.9% at the earlier and later stage, respectively) than in the control (1.0%), the excess being almost entirely due to daughters. Genetic tests of a large number of such offspring failed to find a genetic cause. Instead, it appears that this phenotype may be influenced by factors in the intrauterine environment. It is concluded that shortly after sperm entry, the paternal genome of the zygote is less sensitive than the maternal one to the induction of mutations by ENU.     

A sensitised mutagenesis screen in the mouse to explore the bovine genome: study of muscle characteristics

Meat yield and quality are closely related to muscle development. The muscle characteristics mainly take place during embryonic and postnatal phases. Thus, genetic control of muscle development in early stages represents a significant stake to improve product quality and production efficiency. In bovine, several programmes have been developed to detect quantitative trait loci (QTL) affecting growth, carcass composition or meat quality traits. Such strategy is incontestably very powerful yet extremely cumbersome and costly when dealing with large animals such as ruminants. Furthermore, the fine mapping of the QTL remains a real challenge. Here, we proposed an alternative approach based on chemical mutagenesis in the mouse combined with comparative genomics to identify regions or genes controlling muscle development in cattle. At present, we isolated seven independent mouse lines of high interest. Two lines exhibit a hypermuscular phenotype, and the other five show various skeletomuscular phenotypes. Detailed characterisation of these mouse mutants will give crucial input for the identification and the mapping of genes that control muscular development. Our strategy will provide the opportunity to understand the function and control of genes involved in improvement of animal physiology.     

An insertional mutagenesis system for analyzing the Chinese cabbage genome using Agrobacterium T-DNA

In this study, we applied insertional mutagenesis using Agrobacterium transfer DNA to functionally characterize the gene of Brassica rapa L. ssp. pekinensis. The specific objectives were to: (i) develop and apply a gene tagging system using plasmid rescue and inverse PCR, (ii) select and analyze mutant lines, and (iii) analyze the phenotypic characteristics of mutants. A total of 3,400 insertional mutant lines were obtained from the Chinese cabbage cultivar, ’seoul’, using optimized condition. Plasmid rescue was performed successfully for transgenic plants with multiple T-DNA insertions, and inverse PCR was performed for plants with a single copy. The isolated flanking DNA sequences were blasted against the NCBI database and mapped to a linkage map. We determined the genetic loci in B. rapa with two methods: RFLP using the rescue clones themselves and sequence homology analysis to the B. rapa sequence database by queries of rescued clones sequences. Compared to wild type, the T₁ progenies of mutant lines showed variable phenotypes, including hairless and wrinkled leaves, rosette-type leaves, and chlorosis symptoms. T-DNA inserted mutant lines were the first population that we developed and will be very useful for functional genomics studies of Chinese cabbage.     

Large-scale mutagenesis: yeast genetics in the genome era

The completion of the DNA sequence of the budding yeast Saccharomyces cerevisiae resulted in the identification of a large number of genes. However, the function of most of these genes is not known. One of the best ways to determine gene function is to carry out mutational and phenotypic analysis. In recent years, several approaches have been developed for the mutational analysis of yeast genes on a large scale. These include transposon-based insertional mutagenesis, and systematic deletions using PCR-based approaches. These projects have produced collections of yeast strains and plasmid alleles that can be screened using novel approaches. Analysis of these collections by the scientific community promises to reveal a great deal of biological information about this organism.     

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

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.     

An alternative to radiation hybrid mapping for large-scale genome analysis in barley

The presence of a monosomic gametocidal chromosome (GC) in a barley chromosome addition line of common wheat generates structural aberrations in the barley chromosome as well as in the wheat chromosomes of gametes lacking the GC. A collection of structurally aberrant barley chromosomes is analogous to a panel of radiation hybrid (RH) mapping and is valuable for high-throughput physical mapping. We developed 90 common wheat lines (GC lines) containing aberrant barley 7H chromosomes induced by a gametocidal chromosome, 2C. DNAs isolated from these GC lines provided a panel of 7H chromosomal fragments in a wheat genetic background, comparable with RH mapping panels in mammals. We used this 7H GC panel and the methodology for RH mapping to physically map PCR-based barley markers, SSRs and AFLPs, onto chromosome 7H, relying on polymorphism between the 7H chromosome and the wheat genome. We call this method GC mapping. This study describes a novel adaptation and combination of methods of inducing chromosomal rearrangements to produce physical maps of markers. The advantages of the presented method are similar to RH mapping in that non-polymorphic markers can be used and the mapping panels can be relatively easily obtained. In addition, mapping results are cumulative when using the same mapping set with new markers. The GC lines will be available from the National Bioresources Project-KOMUGI (). Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

2001: a mouse genome odyssey

A report on the 15th International Mouse Genome Conference, Edinburgh, UK, 21-24 October 2001.     

Recent advances in large-scale transposon mutagenesis

Transposons were identified as mobile genetic elements over fifty years ago and subsequently became powerful tools for molecular-genetic studies. Recently, transposon-mutagenesis strategies have been developed to identify essential and pathogenicity-related genes in pathogenic microorganisms. Also, a number of in vitro transposition systems have been used to facilitate genome sequence analysis. Finally, transposon mutagenesis of yeast and complex eukaryotes has provided valuable functional genomic information to complement genome-sequencing projects.     

Analysis of breeding and pathology helps refine management practices of a large-scale N'-ethyl-N'-nitrosourea mouse mutagenesis programme

N'-ethyl-N'-nitrosourea (ENU) is a powerful germline mutagen used in conjunction with phenotype-driven screens to generate novel mouse mutants. ENU also induces genetic lesions in somatic cells and dosage requires optimization between maximum germline mutation rate versus induced sterility and tumourigenesis that compromise the welfare and fecundity of the ENU-treated males. Here, we present our experience with BALB/cAnNCrl and C57BL/6J mice in terms of the pathology induced by ENU and its impact on breeding. In both mouse strains, morbidity and mortality rises with ENU dose. In more than 75% of C57BL/6J males, morbidity and mortality were attributable to the development of malignant T-lymphoblastic lymphoma. Approximately 50% of ENU-treated BALB/cAnNCrl males develop early malignant T-lymphoblastic lymphoma, but the cohort that survives develops late-onset lung carcinoma. Within strains, the latency of these clinically important tumour(s) was not dosage-dependent, but the proportion of mice developing tumours and consequently removed from the breeding programme increased with ENU dosage. The median number of offspring per ENU-treated C57BL/6J male in standard matings with C3H/HeH females decreased with increasing dosage. The two most important underlying causes for lower male fecundity were increased infertility in the highest dosage group and reduced numbers of litters born to the remaining fertile C57BL/6J males due to a higher incidence of morbidity. These findings have allowed us to refine breeding strategy. To maximize the number of offspring from each ENU-treated male, we now rotate productive males between two cages to expose them to more females. This optimizes the number of mutation carrying offspring while reducing the number of ENU-treated males that must be generated.