Mouse genomics

The Mouse genomics team is led by Professor Allan Bradley FRS. Allan is former Director of the Wellcome Trust Sanger Institute, where he holds the title of Director Emeritus.

The decoding of the sequence of the human genome and the genomes of mammalian model organisms like the mouse has revealed that they encode approximately 20,000 genes. However, the role of most of these genes in normal development and physiological processes as well as their function in disease is poorly understood. Discovering the function of each and every gene is fundamental to understanding biological processes in man and other organisms.

[Genome Research Limited]

Background

Professor Bradley's team use a genetic approach to examine gene function. Large-scale disruption of genes in embryonic stem (ES) cells and in somatic tissues of mice is being used to discover genes involved in basic cellular processes as well as diseases like cancer. On a smaller scale mice are being made with mutations in individual genes to investigate their function throughout development and in adult life.

Research

Our aims

The Mouse genomics team at the Wellcome Trust Sanger Institute has two major aims:

The first, which we have pursued for more than two decades, is to develop genetic technologies that enhance our ability to perform genetic analysis of the mouse genome.

The second has been to deploy these technologies to investigate the biological function of genes by generating and analysing mutations in the mouse germ line, in somatic tissues or in embryonic stem cells in culture.

Figure 1: microinjection of embryonic stem cells into a 3.5 day blastocyst, viewed under a microscope.

Figure 1: microinjection of embryonic stem cells into a 3.5 day blastocyst, viewed under a microscope.

Our approach

The isolation of embryonic stem cells and the discovery that these cells can colonise the germ line of other mice following microinjection into pre-implantation blastocysts [Figure 1] established a route to modify the mouse genome. We have devised efficient methods to generate mutations in embryonic stem cells, ranging from point mutations in single genes to the engineering of multi-megabase deletions, duplications and chromosomal inversions.

We are using these chromosome engineering and gene knockout techniques in mouse embryonic stem cells.

One of the genetic platforms used by the group are embryonic stem cells and mice deficient for the Blm gene (Luo et al., 2000). We generated Blm-knockout mice shortly after the Bloom syndrome gene was identified in humans. Although Blm-deficient mice show many of the features of the human disease, we are particularly interested in the use of Blm-deficient cells as a genetic tool. Somatic cells in Blm knockout mice have an unusual tendency to convert, at a very high frequency, any mutation which is heterozygous into homozygous mutations. Blm-deficient ES cells have this same characteristic which we have exploited to generate "libraries" of ES cells which have homozygous mutations in various genes.

Genetic screens using these libraries has enabled us to identify genes required for DNA mismatch repair (MMR) a system that detects and repairs DNA damage (Guo et al., 2004). We have also recently identified mutations in genes which interfere with retroviral infection, such genes are potentially important targets for pharmaceutical intervention in controlling diseases caused by retroviruses (Wang et al., 2007). Over the last year we have used the PiggyBac transposon to generate second generation libraries with deep unbiased genome coverage. Using these libraries we have identified new host genes required by retroviruses for infection and toxicity mediated by ricin.

The laboratory also conducts genetic screens in somatic cells in vivo. These screens are directed towards the identification of tumour suppressor genes and also take advantage of the Blm-deficient genetic background and PiggyBac mutagenesis. In pursuit of this objective we have established and validated a series of transgenic mouse lines with PiggyBac transposon arrays on several different chromosomes which can be used with tissue and temporally regulated transposase expression to affect loss and gain of function somatic mutagenesis.

In addition to performing genome-wide screens we continue to investigate the role of individual genes. The characteristics of the mouse mutants can rarely be accurately predicted. One recent example was the analysis of the function of the microRNA gene, Bic (Rodriguez et al., 2007). Surprisingly mice which had this gene knocked out had an immune deficiency revealing that the gene encoding this particular control molecule has a major role in immune function.

We are also using mice to identify the gene(s) responsible for human deletion syndromes. We have been engaged in the analysis of human chromosome 17 for several years. By making mice with the same deletion as found in human patients with developmental defects affecting the heart and oesophagus we were able to model aspects of the human disease (Yu et al., 2006). This provides an excellent starting point to genetically dissect the region by making smaller deletions which will eventually enable us to identify the relevant gene(s).

Transposons for insertional mutagenesis and induction of stem cells

Pluripotent stem cells can be induced from mouse somatic cells by a novel genetic system that introduces reprogramming factors which can then be removed with no footprint, following research carried out by Allan Bradley’s group at the Sanger Institute (Yusa et al. 2009). The piggyBac transposon, previously developed for insertional mutagenesis, carries 2A peptide-linked reprogramming factors which induce reprogramming of mouse embryonic fibroblasts with efficiencies equivalent to retroviral transduction. The transposon has two advantages, first all of the re-programming factors can be delivered in a single transposon and this can then be excised from the genome without leaving a trace, and thus the induced stem cells are clean of potentially mutagenic sequences and have potential for therapeutic use.

The team has also recently constructed a library composed of 14,000 individual gene-trap clones using piggyBac transposon-mediated mutagenesis in Blm-deficient mouse embryonic stem cells. A genetic screen conducted using this library identified cells with defects in DNA mismatch repair genes. Independent mutations in all known genes of the pathway Msh2, Msh6, Pms2, and Mlh1 were recovered in these screens. The genomic coverage in this library confirms its utility as a new genetic resource for conducting recessive genetic screens in mammalian cells (Wang et al. 2009).

Selected Publications

  • Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon.

    Yusa K, Rad R, Takeda J and Bradley A

    Nature methods 2009;6;5;363-9

  • A piggyBac transposon-based genome-wide library of insertionally mutated Blm-deficient murine ES cells.

    Wang W, Bradley A and Huang Y

    Genome research 2009;19;4;667-73

  • Requirement of bic/microRNA-155 for normal immune function.

    Rodriguez A, Vigorito E, Clare S, Warren MV, Couttet P, Soond DR, van Dongen S, Grocock RJ, Das PP, Miska EA, Vetrie D, Okkenhaug K, Enright AJ, Dougan G, Turner M and Bradley A

    Science (New York, N.Y.) 2007;316;5824;608-11

  • A recessive genetic screen for host factors required for retroviral infection in a library of insertionally mutated Blm-deficient embryonic stem cells.

    Wang W and Bradley A

    Genome biology 2007;8;4;R48

  • A deficiency in the region homologous to human 17q21.33-q23.2 causes heart defects in mice.

    Yu YE, Morishima M, Pao A, Wang DY, Wen XY, Baldini A and Bradley A

    Genetics 2006;173;1;297-307

  • Mismatch repair genes identified using genetic screens in Blm-deficient embryonic stem cells.

    Guo G, Wang W and Bradley A

    Nature 2004;429;6994;891-5

  • Cancer predisposition caused by elevated mitotic recombination in Bloom mice.

    Luo G, Santoro IM, McDaniel LD, Nishijima I, Mills M, Youssoufian H, Vogel H, Schultz RA and Bradley A

    Nature genetics 2000;26;4;424-9

Team

Team members

Caitlin Stewart

cs19@sanger.ac.uk PhD Student

Undertook a BSc in Biological Sciences (Molecular Genetics) at the University of Edinburgh before joining the Sanger Institute.

Research

The title of my PhD Thesis project is "Genetic screens in murine haploid embryonic stem cell lines".

* quick link - http://q.sanger.ac.uk/mousegen