Genomic segmental polymorphisms in inbred mouse strains

By analyzing genomic copy-number differences using high-resolution mouse whole-genome BAC arrays, we uncover substantial differences in regional DNA content between inbred strains of mice. The identification of these apparently common segmental polymorphisms suggests that these differences can contribute to genetic variability and pathologic susceptibility.     

Mitotic recombination is suppressed by chromosomal divergence in hybrids of distantly related mouse strains

Mitotic recombination occurs with high frequency in humans and mice. It leads to loss of heterozygosity (LOH) at important gene loci and can cause disease. However, the genetic modulators of mitotic recombination are not well understood. As recombination depends on a high level of nucleotide sequence homology, we postulate that the frequency of somatic variants derived from mitotic recombination should be diminished in progeny from crosses between strains of mice in which nucleotide sequences have diverged. Here we report that mitotic recombination is suppressed, to various degrees in different tissues, in hybrids of distantly related mouse strains. Reintroduction of greater chromosomal homology by backcrossing restores mitotic recombination in offspring. Thus, chromosomal divergence inhibits mitotic recombination and, consequently, may act as a modifier of cancer susceptibility by limiting the rate of LOH. The suppression of mitotic recombination in some F1 hybrids in which meiotic recombination persists indicates that these processes are differentially affected by chromosomal divergence.     

Complex haplotypes, copy number polymorphisms and coding variation in two recently divergent mouse strains

Inbred mouse strains provide the foundation for mouse genetics. By selecting for phenotypic features of interest, inbreeding drives genomic evolution and eliminates individual variation, while fixing certain sets of alleles that are responsible for the trait characteristics of the strain. Mouse strains 129Sv (129S5) and C57BL/6J, two of the most widely used inbred lines, diverged from common ancestors within the last century, yet very little is known about the genomic differences between them. By comparative genomic hybridization and sequence analysis of 129S5 short insert libraries, we identified substantial structural variation, a complex fine-scale haplotype pattern with a continuous distribution of diversity blocks, and extensive nucleotide variation, including nonsynonymous coding SNPs and stop codons. Collectively, these genomic changes denote the level and direction of allele fixation that has occurred during inbreeding and provide a basis for defining what makes these mouse strains unique.     

Genome-wide patterns of genetic variation among elite maize inbred lines

We have resequenced a group of six elite maize inbred lines, including the parents of the most productive commercial hybrid in China. This effort uncovered more than 1,000,000 SNPs, 30,000 indel polymorphisms and 101 low-sequence-diversity chromosomal intervals in the maize genome. We also identified several hundred complete genes that show presence/absence variation among these resequenced lines. We discuss the potential roles of complementation of presence/absence variations and other deleterious mutations in contributing to heterosis. High-density SNP and indel polymorphism markers reported here are expected to be a valuable resource for future genetic studies and the molecular breeding of this important crop.     

Candidate lung tumor susceptibility genes identified through whole-genome association analyses in inbred mice

We performed a whole-genome association analysis of lung tumor susceptibility using dense SNP maps ( approximately 1 SNP per 20 kb) in inbred mice. We reproduced the pulmonary adenoma susceptibility 1 (Pas1) locus identified in previous linkage studies and further narrowed this quantitative trait locus (QTL) to a region of less than 0.5 Mb in which at least two genes, Kras2 (Kirsten rat sarcoma oncogene 2) and Casc1 (cancer susceptibility candidate 1; also known as Las1), are strong candidates. Casc1 knockout mouse tumor bioassays showed that Casc1-deficient mice were susceptible to chemical induction of lung tumors. We also found three more genetic loci for lung adenoma development. Analysis of one of these candidate loci identified a previously uncharacterized gene Lasc1, bearing a nonsynonymous substitution (D102E). We found that the Lasc1 Glu102 allele preferentially promotes lung tumor cell growth. Our findings demonstrate the prospects for using dense SNP maps in laboratory mice to refine previous QTL regions and identify genetic determinants of complex traits.     

The knockout mouse project

Mouse knockout technology provides a powerful means of elucidating gene function in vivo, and a publicly available genome-wide collection of mouse knockouts would be significantly enabling for biomedical discovery. To date, published knockouts exist for only about 10% of mouse genes. Furthermore, many of these are limited in utility because they have not been made or phenotyped in standardized ways, and many are not freely available to researchers. It is time to harness new technologies and efficiencies of production to mount a high-throughput international effort to produce and phenotype knockouts for all mouse genes, and place these resources into the public domain.     

Analysing complex genetic traits with chromosome substitution strains

Many valuable animal models of human disease are known and new models are continually being generated in existing inbred strains,. Some disease models are simple mendelian traits, but most have a polygenic basis. The current approach to identifying quantitative trait loci (QTLs) that underlie such traits is to localize them in crosses, construct congenic strains carrying individual QTLs, and finally map and clone the genes. This process is time-consuming and expensive, requiring the genotyping of large crosses and many generations of breeding. Here we describe a different approach in which a panel of chromosome substitution strains (CSSs) is used for QTL mapping. Each of these strains has a single chromosome from the donor strain substituting for the corresponding chromosome in the host strain. We discuss the construction, applications and advantages of CSSs compared with conventional crosses for detecting and analysing QTLs, including those that have weak phenotypic effects.     

Epistatic buffering of fitness loss in yeast double deletion strains

Interactions between deleterious mutations have been insufficiently studied, despite the fact that their strength and direction are critical for understanding the evolution of genetic recombination and the buildup of mutational load in populations. We compiled a list of 758 yeast gene deletions causing growth defects (from the Munich Information Center for Protein Sequences database and ref. 7). Using BY4741 and BY4742 single-deletion strains, we carried out 639 random crosses and assayed growth curves of the resulting progeny. We show that the maximum growth rate averaged over strains lacking deletions and those with double deletions is higher than that of strains with single deletions, indicating a positive epistatic effect. This tendency is shared by genes belonging to a variety of functional classes. Based on our data and former theoretical work, we suggest that epistasis is likely to diminish the negative effects of mutations when the ability to produce biomass at high rates contributes significantly to fitness.     

Genetic analysis of the mouse brain proteome

Proteome analysis is a fundamental step in systematic functional genomics. Here we have resolved 8,767 proteins from the mouse brain proteome by large-gel two-dimensional electrophoresis. We detected 1,324 polymorphic proteins from the European collaborative interspecific backcross. Of these, we mapped 665 proteins genetically and identified 466 proteins by mass spectrometry. Qualitatively polymorphic proteins, to 96%, reflect changes in conformation and/or mass. Quantitatively polymorphic proteins show a high frequency (73%) of allele-specific transmission in codominant heterozygotes. Variations in protein isoforms and protein quantity often mapped to chromosomal positions different from that of the structural gene, indicating that single proteins may act as polygenic traits. Genetic analysis of proteomes may detect the types of polymorphism that are most relevant in disease-association studies.     

A radiation hybrid map of mouse genes

A comprehensive gene-based map of a genome is a powerful tool for genetic studies and is especially useful for the positional cloning and positional candidate approaches. The availability of gene maps for multiple organisms provides the foundation for detailed conserved-orthology maps showing the correspondence between conserved genomic segments. These maps make it possible to use cross-species information in gene hunts and shed light on the evolutionary forces that shape the genome. Here we report a radiation hybrid map of mouse genes, a combined project of the Whitehead Institute/Massachusetts Institute of Technology Center for Genome Research, the Medical Research Council UK Mouse Genome Centre, and the National Center for Biotechnology Information. The map contains 11,109 genes, screened against the T31 RH panel and positioned relative to a reference map containing 2,280 mouse genetic markers. It includes 3,658 genes homologous to the human genome sequence and provides a framework for overlaying the human genome sequence to the mouse and for sequencing the mouse genome.     

Detection of regulatory variation in mouse genes

Functional polymorphism in genes can be classified as coding variation, altering the amino-acid sequence of the encoded protein, or regulatory variation, affecting the level or pattern of expression of the gene. Coding variation can be recognized directly from DNA sequence, and consequently its frequency and characteristics have been extensively described. By contrast, virtually nothing is known about the extent to which gene regulation varies in populations. Yet it is likely that regulatory variants are important in modulating gene function: alterations in gene regulation have been proposed to influence disease susceptibility and to have been the primary substrate for the evolution of species. Here, we report a systematic study to assess the extent of cis-acting regulatory variation in 69 genes across four inbred mouse strains. We find that at least four of these genes show allelic differences in expression level of 1.5-fold or greater, and that some of these differences are tissue specific. The results show that the impact of regulatory variants can be detected at a significant frequency in a genomic survey and suggest that such variation may have important consequences for organismal phenotype and evolution. The results indicate that larger-scale surveys in both mouse and human could identify a substantial number of genes with common regulatory variation.     

Engineering a mouse balancer chromosome.

Balancer chromosomes are genetic reagents that are used in Drosophila melanogaster for stock maintenance and mutagenesis screens. Despite their utility, balancer chromosomes are rarely used in mice because they are difficult to generate using conventional methods. Here we describe the engineering of a mouse balancer chromosome with the Cre-loxP recombination system. The chromosome features a 24-centiMorgan (cM) inversion between Trp53 (also known as p53) and Wnt3 on mouse chromosome 11 that is recessive lethal and dominantly marked with a K14-Agouti transgene. When allelic to a wild-type chromosome, the inversion suppresses crossing over in the inversion interval, accompanied by elevated recombination in the flanking regions. The inversion functions as a balancer chromosome because it can be used to maintain a lethal mutation in the inversion interval as a self-sustaining trans-heterozygous stock. This strategy can be used to generate similar genetic reagents throughout the mouse genome. Engineering of visibly marked inversions and deficiencies is an important step toward functional analyses of the mouse genome and will facilitate large-scale mutagenesis programs.     

Dicer is essential for mouse development

To address the biological function of RNA interference (RNAi)-related pathways in mammals, we disrupted the gene Dicer1 in mice. Loss of Dicer1 lead to lethality early in development, with Dicer1-null embryos depleted of stem cells. Coupled with our inability to generate viable Dicer1-null embryonic stem (ES) cells, this suggests a role for Dicer, and, by implication, the RNAi machinery, in maintaining the stem cell population during early mouse development.     

Current issues in mouse genome engineering

The mouse is the foremost vertebrate experimental model because its genome can be precisely and variously engineered. Now that the mouse genome has been sequenced and annotated, the task of mutating each gene is feasible, and an international cooperation is providing mutated embryonic stem cells and mice as readily available resources. Because these resources will change biomedical research, decisions about their nature will have far-reaching effects. It is therefore timely to consider topical issues for mouse genome engineering, such as the background genotype; homologous, site-specific and transpositional recombination; conditional mutagenesis; RNA-mediated interference; and functional genomics with embryonic stem cells.     

Silencing of unsynapsed meiotic chromosomes in the mouse

In Neurospora, DNA unpaired in meiosis both is silenced and induces silencing of all DNA homologous to it. This process, called meiotic silencing by unpaired DNA, is thought to protect the host genome from invasion by transposable elements. We now show that silencing of unpaired (unsynapsed) chromosome regions also takes place in the mouse during both male and female meiosis. The tumor suppressor protein BRCA1 is implicated in this silencing, mirroring its role in the meiotic silencing of the X and Y chromosomes in normal male meiosis. These findings impact on the interpretation of the relationship between synaptic errors and sterility in mammals and extend our understanding of the biology of Brca1.     

The origins and uses of mouse outbred stocks

Outbred mouse stocks, often used in genetics, toxicology and pharmacology research, have been generated in rather haphazard ways. Understanding the characteristics of these stocks and their advantages and disadvantages is important for experimental design. In many studies these mice are used inappropriately, wasting animals' lives and resources on suboptimal experiments. Recently, however, researchers from the field of complex trait analysis have capitalized on the genetics of outbred stocks to refine the identification of quantitative trait loci. Here we assess the most widely used outbred stocks of mice and present guidelines for their use.     

A mouse model of human L1 retrotransposition

The L1 retrotransposon has had an immense impact on the size and structure of the human genome through a variety of mechanisms, including insertional mutagenesis. To study retrotransposition in a living organism, we created a mouse model of human L1 retrotransposition. Here we show that L1 elements can retrotranspose in male germ cells, and that expression of a human L1 element under the control of its endogenous promoter is restricted to testis and ovary. In the mouse line with the highest level of L1 expression, we found two de novo L1 insertions in 135 offspring. Both insertions were structurally indistinguishable from natural endogenous insertions. This suggests that an individual L1 element can have substantial mutagenic potential. In addition to providing a valuable in vivo model of retrotransposition in mammals, these mice are an important step in the development of a new random mutagenesis system.