Novel lethal mouse mutants produced in balancer chromosome screens

Mutagenesis screens are a valuable method to identify genes that are required for normal development. Previous mouse mutagenesis screens for lethal mutations were targeted at specific time points or for developmental processes. Here we present the results of lethal mutant isolation from two mutagenesis screens that use balancer chromosomes. One screen was localized to mouse chromosome 4, between the STS markers D4Mit281 and D4Mit51. The second screen covered the region between Trp53 and Wnt3 on mouse chromosome 11. These screens identified all lethal mutations in the balancer regions, without bias towards any phenotype or stage of death. We have isolated 19 lethal lines on mouse chromosome 4, and 59 lethal lines on chromosome 11, many of which are distinct from previous mutants that map to these regions of the genome. We have characterized the mutant lines to determine the time of death, and performed a pair-wise complementation cross to determine if the mutations are allelic. Our data suggest that the majority of mouse lethal mutations die during mid-gestation, after uterine implantation, with a variety of defects in gastrulation, heart, neural tube, vascular, or placental development. This initial group of mutants provides a functional annotation of mouse chromosomes 4 and 11, and indicates that many novel developmental phenotypes can be quickly isolated in defined genomic intervals through balancer chromosome mutagenesis screens.     

Two new balancer chromosomes on mouse chromosome 4 to facilitate functional annotation of human chromosome 1p

To facilitate genetic screens to identify and maintain recessive mutations that map to the short arm of human chromosome 1, we have utilized chromosome engineering to generate two mouse strains that carry large inversions on the distal region of mouse chromosome 4. The inversion intervals are 16 and 22 cM in size together they cover approximately half of chromosome 4. Since recombination between the wild-type and inversion chromosomes does not occur within these inversion intervals, mutant alleles of genes mapping to this region can be identified and maintained. Therefore, these inversion chromosomes work as balancer chromosomes. These inversions have the additional advantage that they are tagged with genes encoding the visible coat color markers tyrosinase and agouti, and therefore the dosage of the inversion chromosome (+/+, Inv/+, Inv/Inv) can be visually recognized. These inversion strains will be extremely useful for mutagenesis screens that focus on functional annotation of human chromosome 1p.     

Two new mouse chromosome 11 balancers

Segmental inversions causing recombination suppression are an essential feature of balancer chromosomes. Meiotic crossing over between homologous chromosomes within an inversion interval will lead to nonviable gametes, while gametes generated from recombination events elsewhere on the chromosome will be unaffected. This apparent recombination suppression has been widely exploited in genetic studies in Drosophila to maintain and analyze stocks carrying recessive lethal mutations. Balancers are particularly useful in mutagenesis screens since they help to establish the approximate genomic location of alleles of genes causing phenotypes. Using the Cre-loxP recombination system, we have constructed two mouse balancer chromosomes carrying 8- and 30-cM inversions between Wnt3 and D11Mit69 and between Trp53 and EgfR loci, respectively. The Wnt3-D11Mit69 inversion mutates the Wnt3 locus and is therefore homozygous lethal. The Trp53-EgfR inversion is homozygous viable, since the EgfR locus is intact and mutations in p53 are homozygous viable. A dominantly acting K14-agouti minigene tags both rearrangements, which enables these balancer chromosomes to be visibly tracked in mouse stocks. With the addition of these balancers to the previously reported Trp53-Wnt3 balancer, most of mouse chromosome 11 is now available in balancer stocks.     

Modification of an existing chromosomal inversion to engineer a balancer for mouse chromosome 15

Chromosomal inversions are valuable genetic tools for mutagenesis screens, where appropriately marked inversions can be used as balancer chromosomes to recover and maintain mutations in the corresponding chromosomal region. For any inversion to be effective as a balancer, it should exhibit both dominant and recessive visible traits; ideally the recessive trait should be a fully penetrant lethality in which inversion homozygotes die before birth. Unfortunately, most inversions recovered by classical radiation or chemical mutagenesis techniques do not have an overt phenotype in either the heterozygous or the homozygous state. However, they can be modified by relatively simple procedures to make them suitable as an appropriately marked balancer. We have used homologous recombination to modify, in embryonic stem cells, the recessive-lethal In(15)21Rk inversion to endow it with a dominant-visible phenotype. Several ES cell lines were derived from inversion heterozygotes, and a keratin-14 (K14) promoter-driven agouti minigene was introduced onto the inverted chromosome 15 in the ES cells by gene targeting. Mice derived from the targeted ES cells carry the inverted chromosome 15 and, at the same time, exhibit lighter coat color on their ears and tails, making this modified In(15)21Rk useful as a balancer for proximal mouse chromosome 15.     

Introducing defined chromosomal rearrangements into the mouse genome

Chromosomal rearrangements have been instrumental in genetic studies in Drosophila. Visibly marked deficiencies (deletions) are used in mapping studies and region-specific mutagenesis screens by providing segmental haploidy required to uncover recessive mutations. Marked recessive lethal inversions are used as balancer chromosomes to maintain recessive lethal mutations and to maintain the integrity of mutagenized chromosomes. In mice, studies on series of radiation-induced deletions that surround several visible mutations have yielded invaluable functional genomic information in the regions analyzed. However, most regions of the mouse genome are not accessible to such analyses due to a lack of marked chromosomal rearrangements. Here we describe a method to generate defined chromosomal rearrangements using the Cre--loxP recombination system based on a published strategy [R. Ramirez-Solis, P. Liu, and A. Bradley, (1995) Nature 378, 720--724]. Various types of rearrangements, such as deletions, duplications, inversions, and translocations, can be engineered using this strategy. Furthermore, the rearrangements can be visibly marked with coat color genes, providing essential reagents for large-scale recessive genetic screens in the mouse. The ability to generate marked chromosomal rearrangements will help to elevate the level of manipulative mouse genetics to that of Drosophila genetics.     

N-ethyl-N-nitrosourea mutagenesis: boarding the mouse mutant express.

In the mouse, random mutagenesis with N-ethyl-N-nitrosourea (ENU) has been used since the 1970s in forward mutagenesis screens. However, only in the last decade has ENU mutagenesis been harnessed to generate a myriad of new mouse mutations in large-scale genetic screens and focused, smaller efforts. The development of additional genetic tools, such as balancer chromosomes, refinements in genetic mapping strategies, and evolution of specialized assays, has allowed these screens to achieve new levels of sophistication. The impressive productivity of these screens has led to a deluge of mouse mutants that wait to be harnessed. Here the basic large- and small-scale strategies are described, as are the basics of screen design. Finally, and importantly, this review describes the mechanisms by which such mutants may be accessed now and in the future. Thus, this review should serve both as an overview of the power of forward mutagenesis in the mouse and as a resource for those interested in developing their own screens, adding onto existing efforts, or obtaining specific mouse mutants that have already been generated.     

N-Ethyl-N-Nitrosourea Mutagenesis: Boarding the Mouse Mutant Express

In the mouse, random mutagenesis with N-ethyl-N-nitrosourea (ENU) has been used since the 1970s in forward mutagenesis screens. However, only in the last decade has ENU mutagenesis been harnessed to generate a myriad of new mouse mutations in large-scale genetic screens and focused, smaller efforts. The development of additional genetic tools, such as balancer chromosomes, refinements in genetic mapping strategies, and evolution of specialized assays, has allowed these screens to achieve new levels of sophistication. The impressive productivity of these screens has led to a deluge of mouse mutants that wait to be harnessed. Here the basic large- and small-scale strategies are described, as are the basics of screen design. Finally, and importantly, this review describes the mechanisms by which such mutants may be accessed now and in the future. Thus, this review should serve both as an overview of the power of forward mutagenesis in the mouse and as a resource for those interested in developing their own screens, adding onto existing efforts, or obtaining specific mouse mutants that have already been generated.     

A new strategy for efficient in vivo screening of mutagenized Drosophila embryos

The analysis of mutants is an indispensable approach towards characterizing gene function. Combining several tools of Drosophila genetics, we designed a new strategy for a mutagenesis screen which is fast, easy-to-apply, and cheap. The combination of a cell-specific Gal4 line with an upstream activating sequence-green fluorescent protein (UAS-GFP) allows the in vivo detection of the cells or tissues of interest without the need for fixation and staining. To further simplify and accelerate the screening procedure, we generated recombinant flies that carry the Gal80 transgene in balancer chromosomes. Gal80 inactivates Gal4; and thus prevents GFP-expression during embryonic and postembryonic development in all individuals carrying the balancer chromosomes. This allows for an easy distinction in vivo between heterozygous and homozygous mutants, the latter being the only ones expressing GFP. Since most of the fly strains and balancer chromosomes can be substituted, this method is suitable for nearly any mutagenesis screen that does not have major restrictions.     

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.     

Regional variation in the density of essential genes in mice

In most species, and particularly in vertebrates, the percentage of genes absolutely required for survival, the essential genes, has not been estimated. To obtain this estimation, we used the mouse as an experimental model to carry out high-efficiency N-ethyl-N-nitrosourea (ENU) mutagenesis screens in two balancer chromosome regions, and compared our results to a third previously published screen. The number of essential genes in each region was predicted based on allele frequencies. We determined that the density of essential genes differs by up to an order of magnitude among genomic regions. This indicates that extrapolating from regional estimates to genome-wide estimates of essential genes has a huge variance. A particularly high density of essential genes on mouse Chromosome 11 coincides with a high degree of regional linkage conservation, providing a possible causal explanation for the density variation. This is the first demonstration of regional variation in essential gene density in the mouse genome.     

Engineering chromosomal rearrangements in mice

The combination of gene-targeting techniques in mouse embryonic stem cells and the Cre/loxP site-specific recombination system has resulted in the emergence of chromosomal-engineering technology in mice. This advance has opened up new opportunities for modelling human diseases that are associated with chromosomal rearrangements. It has also led to the generation of visibly marked deletions and balancer chromosomes in mice, which provide essential reagents for maximizing the efficiency of large-scale mutagenesis efforts and which will accelerate the functional annotation of mammalian genomes, including the human genome.     

Retention of induced mutations in a Drosophila reverse-genetic resource

Targeting induced local lesions in genomes (TILLING) is a reverse-genetic method for identifying point mutations in chemically mutagenized populations. For functional genomics, it is ideal to have a stable collection of heavily mutagenized lines that can be screened over an extended period of time. However, long-term storage is impractical for Drosophila, so mutant strains must be maintained by continual propagation of live cultures. Here we evaluate a strategy in which ethylmethane sulfonate (EMS) mutagenized chromosomes were maintained as heterozygotes with balancer chromosomes for >100 generations before screening. The strategy yielded a spectrum of point mutations similar to those found in previous studies of EMS-induced mutations, as well as 2.4% indels (insertions and deletions). Our analysis of 1887 point mutations in 148 targets showed evidence for selection against deleterious lesions and differential retention of lesions among targets on the basis of their position relative to balancer breakpoints, leading to a broad distribution of mutational densities. Despite selection and differential retention, the success of a user-funded service based on screening a large collection several years after mutagenesis indicates sufficient stability for use as a long-term reverse-genetic resource. Our study has implications for the use of balancer chromosomes to maintain mutant lines and provides the first large-scale quantitative assessment of the limitations of using breeding populations for repositories of genetic variability.     

Mutagenesis and phylogenesis of plants

Genetic chromosomes and genome mutations are taken into consideration. For a comparative study of mutagenesis types, data of separate families of seed plants bearing various vital forms of different evolutionary age were used. It was indicated that, in most ancient gymnosperms, the primitive mechanism of genetic mutation has been prevailing. In the process of evolution, chromosome and genome mutagenesis types were added, which enables the creation of new genes without functional disturbances of the ancestral system. Connections between various vital forms and mutagenese types are considered.     

Rodent models of genetic disease

The genetic study of rats and mice using natural variants, natural mutations, chemical or radiation induced mutations, engineered mutations and conditional engineered mutations has provided the tools for investigating the genetics of disease. The completion of the mouse genomic sequence and progress towards sequencing the rat genome in the past year will enable the molecular identification of quantitative trait loci and induced mutations. Sequence-based single nucleotide polymorphism discovery and a greater understanding of the haplotype structure of inbred strains is revitalising quantitative trait locus mapping and there are now plans for an ambitious eight-way recombinant inbred cross and renewed interest in existing resources such as heterogeneous stocks. In the past year there have been refinements to ENU mutagenesis approaches including balancer chromosomes and a new gene-driven approach.     

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.     

Nature of Deleterious Mutation Load in Drosophila

Much population genetics and evolution theory depends on knowledge of genomic mutation rates and distributions of mutation effects for fitness, but most information comes from a few mutation accumulation experiments in Drosophila in which replicated chromosomes are sheltered from natural selection by a balancer chromosome. I show here that data from these experiments imply the existence of a large class of minor viability mutations with approximately equivalent effects. However, analysis of the distribution of viabilities of chromosomes exposed to EMS mutagenesis reveals a qualitatively different distribution of effects lacking such a minor effects class. A possible explanation for this difference is that transposable element insertions, a common class of spontaneous mutation event in Drosophila, frequently generate minor viability effects. This explanation would imply that current estimates of deleterious mutation rates are not generally applicable in evolutionary models, as transposition rates vary widely. Alternatively, much of the apparent decline in viability under spontaneous mutation accumulation could have been nonmutational, perhaps due to selective improvement of balancer chromosomes. This explanation accords well with the data and implies a spontaneous mutation rate for viability two orders of magnitude lower than previously assumed, with most mutation load attributable to major effects.     

Manipulating the <Emphasis Type="Italic">Xenopus</Emphasis>genome with transposable elements

The study of amphibian embryogenesis has provided important insight into the mechanisms of vertebrate development. The frog Xenopus laevis has been an important model of vertebrate cell biology and development for many decades. Genetic studies in this organism are not practical because of the tetraploid nature of the genome and the long generation time of this species. Recently, a closely related frog, namely Xenopus tropicalis, has been proposed as an alternative system; it shares all of the physical characteristics that make X. laevis a useful model but has the advantage of a diploid genome and short generation time. The rapid accumulation of genetic resources for this animal and the success of pilot mutagenesis screens have helped propel this model system forward. Transposable elements will provide invaluable tools for manipulating the frog genome. These integration systems are ideally suited to transgenesis and insertional mutagenesis strategies in the frog. The high fecundity of the frog combined with the ability to remobilize transposon transgenes integrated into frog genome will allow large-scale insertional mutagenesis screens to be performed in laboratories with modest husbandry capacities.     

Chromosome-scale genetic mapping using a set of 16 conditionally stable Saccharomyces cerevisiae chromosomes

We have created a resource to rapidly map genetic traits to specific chromosomes in yeast. This mapping is done using a set of 16 yeast strains each containing a different chromosome with a conditionally functional centromere. Conditional centromere function is achieved by integration of a GAL1 promoter in cis to centromere sequences. We show that the 16 yeast chromosomes can be individually lost in diploid strains, which become hemizygous for the destabilized chromosome. Interestingly, most 2n - 1 strains endoduplicate and become 2n. We also demonstrate how chromosome loss in this set of strains can be used to map both recessive and dominant markers to specific chromosomes. In addition, we show that this method can be used to rapidly validate gene assignments from screens of strain libraries such as the yeast gene disruption collection.     

Long-range autocorrelations of CpG islands in the human genome

In this paper, we use a statistical estimator developed in astrophysics to study the distribution and organization of features of the human genome. Using the human reference sequence we quantify the global distribution of CpG islands (CGI) in each chromosome and demonstrate that the organization of the CGI across a chromosome is non-random, exhibits surprisingly long range correlations (10 Mb) and varies significantly among chromosomes. These correlations of CGI summarize functional properties of the genome that are not captured when considering variation in any particular separate (and local) feature. The demonstration of the proposed methods to quantify the organization of CGI in the human genome forms the basis of future studies. The most illuminating of these will assess the potential impact on phenotypic variation of inter-individual variation in the organization of the functional features of the genome within and among chromosomes, and among individuals for particular chromosomes.