Phenotype-driven mutagenesis has provided a new tool that will help discover the function of genes. Rudi Balling discusses the role of mutant mice in functional genomics
This year marks the completion of one of the largest biological projects that scientists have ever undertaken: sequencing the human genome. But it does not mark the completion of the Human Genome Project.
Although the availability of the sequence of 3 billion base pairs of human DNA is a remarkable milestone in science, most of the challenges of the genome project are only just beginning. The reason for this can be paraphrased in a statement that Sidney Bremer made recently: "There is difference between information and knowledge."
Even if we know the order of 3 billion base pairs and even if we know where along these 3 billion each of the estimated 35,000-40,000 genes starts and ends, we still do not know what each of these genes does, and when, where and to which level it is expressed, nor how it responds when the organism takes in food, drugs or exercise or is infected by a virus or bacterium.
Such experiments, which address the function of genes within the entire genome, are at the centre of research at the GSF-Institute of Mammalian Genetics in Munich. The most direct and efficient way of addressing the function of genes is by removing them from an organism. This, of course, cannot be done in humans. This is why we use mice as a model organism.
Over the past ten years, it has become routine to add or remove genes from the genome of mice, either by injecting DNA into the pronucleus of one-cell mouse embryos or by gene-targeting techniques, which take advantage of the mechanism of homologues recombination. Using pluripotent embryonic stem cells for genetic manipulation in the Petri dish and then reintroducing these cells into embryos by making chimeras has opened a new toolbox for analysing the role of genes during development, in the pathogenesis of disease and in the response to environmental factors.
During the past five years, our institute has added a new tool to functional genomics: producing mutants by chemical mutagenesis and selecting interesting mutants among the offspring of the mutagenised mice for interesting diseases or other deviant phenotypes. This is called phenotype-driven mutagenesis, compared with the gene-driven mutagenesis strategy that is used in knockout or transgenic experiments. How does this process work?
Male mice are injected with ENU (Ethylnitrosurea), a powerful mutagen that induces point mutations in the DNA. Importantly, these mutations are induced in the spermatogonia of mice, so that offspring from mutagenised mice inherit the mutations and the corresponding diseases that are caused by these mutations. One can either screen for dominant mutations in the first generation or for recessive diseases if one raises families (or pedigrees) of these offspring.
Two points are important: first, the phenotypic assay used to screen mutagenised mice determines what kind of mutants will be recovered. A general rule is, you find what you look for. Second, with the identification of a mutant, one does not have the gene that causes the disease in hand. Backcross strategies, to map chromosomally the mutation followed by positional or candidate cloning need to follow the first step of mutant identification. This is again different from knockout experiments, where one has the gene first but then tries to identify a phenotype or disease that develops if this gene is missing. In one case, you have a gene in search of a phenotype, in the second, a phenotype in search of a gene.
The GSF-Institute of Mammalian Genetics has screened more than 25,000 mice for dominant mutations over four years. We have isolated more than 250 new mouse mutants, which successfully pass on their phenotypic abnormality from generation to generation.
What kind of mutations were recovered? Of course the easiest ones are those that can be seen with the naked eye. These include mice with kinky tails, changed coat colours, duplicated fingers or with abnormal behaviour such as running in a circle.
More sophisticated methods have been introduced recently to identify mice that cannot hear, that have high blood glucose or cholesterol levels or different percentages of B or T cells. New assays are set up to identify mice that have reduced bone density (osteoporosis) or an increased or decreased susceptibility to bacterial infection.
Two major large-scale ENU mutagenesis projects have been launched worldwide. One at the GSF Research Center in Munich, the second at the MRC Genetic Unit in Harwell, England. After proof of concept was established by these two projects, many countries have embarked on phenotype-driven mutagenesis. Three centres will be founded in the United States by the National Institutes of Health, one in Japan and another one in Australia.
The future bottleneck in functional genomics, however, will not be the production of mutants. It will be the detailed phenotypic characterisation of mutants combined with the efficient cloning and molecular characterisation of mice.
"Treasure the odds" is a saying among geneticists. "Treasure the mutants" is the motto of the ENU projects.
Rudi Balling, Institute of Mammalian Genetics, GSF Research Center for Environment and Health Neuherberg, Germany.