Our consortium uses forward genetic methods to identify genes that are essential for the mammalian immune response, and to determine their functions relative to one another. The current approach entails the induction of thousands of random germline point mutations on a defined genetic background (C57BL/6) using N-ethyl-N-nitrosourea (ENU), the phenotypic screening of many thousands of mice for specific defects of immunity, and the positional cloning of those transmissible mutations that are detected. Over time, the effects of millions of point mutations that affect coding sense may be probed. In terms of throughput, the ENU mutagenesis effort now underway is among the largest in the world, and presently the only one primarily devoted to the deciphering of innate immunity. The list of transmissible mutations is available on the Mouse Mutants web site. For more detailed description of these studies see our forward genetics page.
Forward Genetics
Forward genetics, coupled with ENU mutagenesis, provides an
unbiased method to identify genes based on observed phenotypes. This method, coupled with well-designed screening
assays, has led to the identification of many genes with previously unknown immunological functions.
Screens may be designed to reveal genes that confer either susceptibility or resistance to infectious challenge.
In addition to revealing unknown genes, ENU mutagenesis can also reveal novel functions for known genes. Because it is a point mutagen, ENU creates many different shades of phenotypic change: null mutations, hypomorphic mutations, hypermorphic mutations, and neomorphic mutations have all been reported. Where a knockout allele may cause embryonic lethality, it is commonly observed that alleles produced by ENU can be viable and informative. Indeed, it is likely that all genes have viable variant alleles.
ENU mutagenesis offers additional advantages over knockout mice. ENU produces mutations on a defined genetic background; by contrast, most knockout mice are produced on a mixed background and even after extensive backcrossing to a defined strain, the congenic interval that contains the knockout mutation also contains a large number of other mutations with undefined effects. Moreover, knockout and gene trap alleles can (and often do) influence the behavior of neighboring loci. For these reasons, ENU mutant mice provide better raw material than knockout mutations for systems biology studies, including microarray and proteomic analysis. Each center for mutagenesis has an impressive track record in the production and isolation of germ-line mutations affecting various aspects of immunity. The TSRI and ANU facilities each currently produce far more germline mutations than any other ENU mutagenesis facilities in the world. They are also the only two ENU mutagenesis programs in the world that focus entirely on immunological phenotypes.
At TSRI, more than 87,000 germline mutant mice have been produced and subjected to various screens. Among these are 62,000 G3 mice, derived from more than 7,000 G1 animals. These have collectively sustained more than 100,000 homozygous coding changes compatible with survival to the age of weaning. The full list of available mutants may be found at mutagenetix.scripps.edu.
At ANU, several thousand pedigrees of G3 animals have been systematically screened for important immunological traits including: flow cytometric screens of lymphocyte subsets; induced antibody responses to Bordetella pertussis, haptenated proteins, and polysaccharides; immunohistological organization of lymphoid tissues; susceptibility to systemic lupus; and susceptibility to type 1 diabetes. To date, the ANU group and its collaborators have mapped 49 new strains to specific chromosomal intervals and identified the mutated gene in 74% of these. These have revealed entirely new pathways and genes, such as the Roquin gene which regulates autoimmunity, follicular helper T cell formation, and ICOS expression at the level of mRNA stability.