Mouse Genetics
Genetic Dissection Of Blood Cell Production
Genetics has been successfully employed in lower organisms including yeast, nematodes, insects and fish to unravel the molecular regulation of complex processes such as development, cell division and cell death. Two factors have combined to make large-scale mouse genetics feasible and accessible to even small laboratories; (1) the demonstration in the early 1980s that ENU is a powerful germ cell mutagen in the mouse and (2) the completion early this century of the mouse genome project.
Mouse mutation identification
Unlike many large national mouse mutagenesis centres, which have broad programs examining many different phenotypes, our approach has been to focus almost exclusively on the haematopoietic system. We have also borrowed a number of tricks, (e.g. modifier and sensitised screens) developed by geneticists using lower organisms such as yeast. Rather than beginning with wild type mice and identifying mutants with an aberrant phenotype, modifier screens commence with a mouse with a pre-existing phenotype and aim to identify mutations that make this phenotype more severe (enhancers) or less severe (suppressors). We are one of the first laboratories to employ this strategy in vertebrates and are particularly excited about this approach because, not only may it shed light on the in vivo regulation of complex biological such haematopoiesis, but also because of its potential to pin-point new targets for the treatment of human disease. Just as most ENU-induced mutations cause loss of function, most small molecule therapies also reduce the function of proteins to which they bind. Accordingly, screens for genes that lead to amelioration of disease when mutated should provide genome-wide access to novel in vivo validated targets for pharmaceutical discovery.
In collaboration with the laboratories of Professor Warren Alexander in the Division of Cancer and Haematology and Dr Benjamin Kile in this Division, our focus has been on dissecting platelet formation; a process that includes elements common to other blood cell lineages, including self-renewal of haematopoietic stem cells and lineage commitment, as well as unique events like polyploidization via endoreduplication, pro-platelet formation and platelet shedding. Many of our studies have commenced with a mouse model of human inherited thrombocytopenia, in which the gene encoding the thrombopoietin receptor, Mpl, has been mutated by homologous recombination in ES cells. Mpl-/- mice have approximately 10% of the normal number of platelets and a corresponding decrease in megakaryocytes, megacaryocyte progenitors and stem cells. Despite their stem cell deficit, Mpl-/- mice have normal numbers of other peripheral blood cells, implying the presence of a compensation mechanism within the haematopoietic system. We have used Mpl-/- in an ENU mutagensis screen to identify suppressors of thrombocytopenia and to find enhancers of the stem cell phenotype that manifest as a multi-lineage defect in the peripheral blood. In addition, we have carried out a conventional ENU mutagenesis screen using Mpl+/+ wild type mice and identified pedigrees with thrombocytopenia and thrombocytosis. To date we have identified more than 30 mutant pedigrees with defects in stem cells and/or the platelet lineage. The causative mutation in 12 of these has been identified and in an additional 10 cases genes in the candidate interval are currently being sequenced. These pedigrees are being intensively studied within our laboratory at both the biological and molecular level.