Tony Cox

The Ensembl genome database project

The Ensembl (http://www.ensembl.org/) database project provides a bioinformatics framework to organise biology around the sequences of large genomes. It is a comprehensive source of stable automatic annotation of the human genome sequence, with confirmed gene predictions that have been integrated with external data sources, and is available as either an interactive web site or as flat files. It is also an open source software engineering project to develop a portable system able to handle very large genomes and associated requirements from sequence analysis to data storage and visualisation. The Ensembl site is one of the leading sources of human genome sequence annotation and provided much of the analysis for publication by the international human genome project of the draft genome. The Ensembl system is being installed around the world in both companies and academic sites on machines ranging from supercomputers to laptops.

DNA methylation profiling of human chromosomes 6, 20 and 22

DNA methylation is the most stable type of epigenetic modification modulating the transcriptional plasticity of mammalian genomes. Using bisulfite DNA sequencing, we report high-resolution methylation profiles of human chromosomes 6, 20 and 22, providing a resource of about 1.9 million CpG methylation values derived from 12 different tissues. Analysis of six annotation categories showed that evolutionarily conserved regions are the predominant sites for differential DNA methylation and that a core region surrounding the transcriptional start site is an informative surrogate for promoter methylation. We find that 17% of the 873 analyzed genes are differentially methylated in their 5′ UTRs and that about one-third of the differentially methylated 5′ UTRs are inversely correlated with transcription. Despite the fact that our study controlled for factors reported to affect DNA methylation such as sex and age, we did not find any significant attributable effects. Our data suggest DNA methylation to be ontogenetically more stable than previously thought.

A conditional knockout resource for the genome-wide study of mouse gene function

Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.

A physical map of the mouse genome

A physical map of a genome is an essential guide for navigation, allowing the location of any gene or other landmark in the chromosomal DNA. We have constructed a physical map of the mouse genome that contains 296 contigs of overlapping bacterial clones and 16,992 unique markers. The mouse contigs were aligned to the human genome sequence on the basis of 51,486 homology matches, thus enabling use of the conserved synteny (correspondence between chromosome blocks) of the two genomes to accelerate construction of the mouse map. The map provides a framework for assembly of whole-genome shotgun sequence data, and a tile path of clones for generation of the reference sequence. Definition of the human–mouse alignment at this level of resolution enables identification of a mouse clone that corresponds to almost any position in the human genome. The human sequence may be used to facilitate construction of other mammalian genome maps using the same strategy.

Chromosomal transposition of PiggyBac in mouse embryonic stem cells

Transposon systems are widely used for generating mutations in various model organisms. PiggyBac (PB) has recently been shown to transpose efficiently in the mouse germ line and other mammalian cell lines. To facilitate PBs application in mammalian genetics, we characterized the properties of the PB transposon in mouse embryonic stem (ES) cells. We first measured the transposition efficiencies of PB transposon in mouse embryonic stem cells. We next constructed a PB/SB hybrid transposon to compare PB and Sleeping Beauty (SB) transposon systems and demonstrated that PB transposition was inhibited by DNA methylation. The excision and reintegration rates of a single PB from two independent genomic loci were measured and its ability to mutate genes with gene trap cassettes was tested. We examined PBs integration site distribution in the mouse genome and found that PB transposition exhibited local hopping. The comprehensive information from this study should facilitate further exploration of the potential of PB and SB DNA transposons in mammalian genetics.

Ensembl 2002: accommodating comparative genomics

The Ensembl (http://www.ensembl.org/) database project provides a bioinformatics framework to organise biology around the sequences of large genomes. It is a comprehensive source of stable automatic annotation of human, mouse and other genome sequences, available as either an interactive web site or as flat files. Ensembl also integrates manually annotated gene structures from external sources where available. As well as being one of the leading sources of genome annotation, Ensembl is an open source software engineering project to develop a portable system able to handle very large genomes and associated requirements. These range from sequence analysis to data storage and visualisation and installations exist around the world in both companies and at academic sites. With both human and mouse genome sequences available and more vertebrate sequences to follow, many of the recent developments in Ensembl have focusing on developing automatic comparative genome analysis and visualisation.

Large-Scale Mutagenesis in p19ARF- and p53-Deficient Mice Identifies Cancer Genes and Their Collaborative Networks

Summary p53 and p19ARF are tumor suppressors frequently mutated in human tumors. In a high-throughput screen in mice for mutations collaborating with either p53 or p19ARF deficiency, we identified 10,806 retroviral insertion sites, implicating over 300 loci in tumorigenesis. This dataset reveals 20 genes that are specifically mutated in either p19ARF-deficient, p53-deficient or wild-type mice (including Flt3, mmu-mir-106a-363, Smg6, and Ccnd3), as well as networks of significant collaborative and mutually exclusive interactions between cancer genes. Furthermore, we found candidate tumor suppressor genes, as well as distinct clusters of insertions within genes like Flt3 and Notch1 that induce mutants with different spectra of genetic interactions. Cross species comparative analysis with aCGH data of human cancer cell lines revealed known and candidate oncogenes (Mmp13, Slamf6, and Rreb1) and tumor suppressors (Wwox and Arfrp2). This dataset should prove to be a rich resource for the study of genetic interactions that underlie tumorigenesis.

Mutagenic insertion and chromosome engineering resource (MICER).

Embryonic stem cell technology revolutionized biology by providing a means to assess mammalian gene function in vivo. Although it is now routine to generate mice from embryonic stem cells, one of the principal methods used to create mutations, gene targeting, is a cumbersome process. Here we describe the indexing of 93,960 ready-made insertional targeting vectors from two libraries. 5,925 of these vectors can be used directly to inactivate genes with an average targeting efficiency of 28%. Combinations of vectors from the two libraries can be used to disrupt both alleles of a gene or engineer larger genomic changes such as deletions, duplications, translocations or inversions. These indexed vectors constitute a public resource (Mutagenic Insertion and Chromosome Engineering Resource; MICER) for high-throughput, targeted manipulation of the mouse genome.

A systematic library for comprehensive overexpression screens in Saccharomyces cerevisiae

Modern genetic analysis requires the development of new resources to systematically explore gene function in vivo. Overexpression screens are a powerful method to investigate genetic pathways, but the goal of routine and comprehensive overexpression screens has been hampered by the lack of systematic libraries. Here we describe the construction of a systematic collection of the Saccharomyces cerevisiae genome in a high-copy vector and its validation in two overexpression screens.

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.