Mutagenesis of the Mouse Genome
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The International Knock-out Mouse Project is a worldwide effort to generate knock-out mutations in every gene in the mouse genome — and is widely considered to be the next most important step following the Human Genome Project. As a major contributor to the International Knock-out Mouse Project resource, this project aims to identify and characterize the functions of mouse genes. The team led by Drs. Hicks and Rossant will establish cell lines in which all mouse genes of interest are knocked out and will make them available to the scientific and biotech communities.
In addition, the team will establish cell-based and computer-based applications linked to target genes associated with disease, and will establish a distribution centre making so that Canadian biomedical research projects, whether public or private, can more easily benefit from this knowledge base. The genetically altered cell lines developed by this project will allow researchers to address the exact role of genetic changes in the development of specific human diseases.
This in turn is expected to accelerate the rate at which new medical discoveries are translated into possible therapeutic interventions, and then moved into health care delivery. These exist in a heterozygous form and do not necessarily cause a phenotype. In our experience, the majority of ENU-induced mutations, behave as recessive traits or are codominant at best.
These G1 animals are then either used for phenotypic screens or can be used to produce G2 mice, which in turn are backcrossed to the G1 male to generate G3 offspring. While screening the G1 population for phenotypes is limited to the identification of dominant mutations, screening of G3 mice allows for the discovery of recessive mutations.
The rate-limiting step in ENU mutagenesis has long been the identification of causative mutations. Until recently, identified mutant lines were outcrossed to genetically different inbred strains and often the analyses of hundreds if not thousands of meiosis were needed to obtain a small enough critical region that could be sequenced. However, the availability of NGS has significantly facilitated the process of variant identification. Currently, targeted exon-enrichment—i. In addition, the availability of NGS also provides further opportunities for the phenotypic probing of ENU germline mutants.
Often, phenotypes identified in ENU germline mice are lost or significantly influenced by modifier loci located on outcross strains carrying a high degree of genetic variation. By being able to analyze and sequence large genomic regions, fine mapping is superfluous and the exploration of subtle phenotypes can be traced following an outcross to strains with minimal genetic variation between the outcross and parent ENU strain. As referred to above, a critical aspect of ENU mutagenesis is the biological field of interest to be probed.
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ENU mutagenesis has been used to define the genetic footprint of a wide variety of phenotypes, including visible, behavioral, developmental and immunological phenotypes 7. Nonetheless, its success is depending on: 1 the use of reliable screening assays with limited biological variation, 2 targeting large genomic footprints, and 3 probing a biological phenotype that is poorly defined. Our laboratory has used ENU mutagenesis to identify genes with non-redundant function in lymphocyte development, priming or effector function. Importantly, the induction of type I or II IFNs are essential for the generation of antigen-specific T cell responses mediated via cell-death induced immune responses 8.
The pathways by which such nucleotide structures drive type I IFN production following administration of apoptotic cells remains still elusive to date. Thus, the in vivo cytotoxicity screen performed in our laboratory presents a large genetic footprint, not only comprising lymphocyte development but also targeting NK-, DC- and T cell biological function.
Using N-ethyl-N-nitrosourea ENU germline mutagenesis, our laboratory previously identified Gimap5-deficient mice—designated sphinx —that exhibit reduced lymphocyte survival and develop severe colitis around weeks of age This mutation resulted in a G38C amino acid substitution in the predicted GTP-binding domain of Gimap5, destabilizing Gimap5 protein expression Gimap5 is part of the family of Gimap genes which are predominantly expressed in lymphocytes and regulate lymphocyte survival during development and homeostasis Gimap proteins contain a GTP-binding AIG1 homology domain, first identified in disease-resistance genes in higher plants 9, More recent crystallographic studies showed that the Gimap proteins resemble a nucleotide coordination and dimerization mode previously observed for dynamin GTPase—a component essential for the scission and fusion of cellular vesicular compartments such as endosomes at the cell surface or the Golgi apparatus in the cytosol Members of the Gimap family appear to be localized to different subcellular compartments with Gimap5 reported to localize in lysosomes based on studies in human, mouse and rat lymphocytes Overall, the function of these proteins and their role in disease development remain poorly defined.
Genetic aberrancies in Gimap5 have been strongly linked to reduced lymphocyte survival and homeostasis, but importantly have also been associated with autoimmune diseases. Together these observations suggest that, beyond lymphocyte survival, Gimap5 is essential for maintaining immunological tolerance.
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Previous work suggests that impaired lymphocyte survival and consequent lymphopenia may be linked to the loss of immunological tolerance. This process—also referred to as lymphopenia-induced proliferation LIP —is accompanied by marked alterations in T cell phenotype and is linked to auto-immunity Schematic representation of the events causing colitis in Gimap5-deficient mice.
The family of Foxo transcription factors contain 4 members of which three Foxo1, Foxo3 and Foxo4 have overlapping patterns of expression and transcriptional activities In addition, Foxo1, 3 expression has been reported to be essential for Treg cell development and function 41, The regulation of Foxo3 and Foxo4 protein expression appears to occur at the post-transcriptional level, although the exact mechanism underlying the loss of Foxo-expression remains to be determined.
They often present significant challenges in terms of treatment due to the wide variety of immune cells that can be affected. Therefore, besides defining the genetic footprint underlying SCID, a critical challenge lies in obtaining a thorough understanding of the degree of the immunodeficiency presented by specific mutations in genes, including defining the types of immune cells affected and functional aberrancies observed.
Deciphering Mammalian Biology and Disease
The G1 pedigrees of these germline mutants were selected to establish a homozygote colony used for genetic analysis and further phenotypic characterization. The mutation behaved as strictly recessive, in that normal cytolytic effector functions were observed in heterozygote mutant mice. In contrast, an increase in the number of macrophages in the spleen was observed Figure 2b.
Notably, upon necropsy, the liver exhibited white patches at the periphery Figure 2c which upon histological analysis revealed large areas of necrosis and significant hematopoietic infiltrate and inflammation Figure 2 d-e. The resulting F1 offspring were intercrossed to generate a F2 and a total of 24 offspring 6 Lampe2 mutant- and 18 wildtype-phenotypes were analyzed for both phenotype and genotype. The critical region was defined by proximal marker rs at position Among the annotated genes, Hematopoietic protein 1 Hem-1 aka NCK associated protein 1 like or Nckap1l presented a clear candidate gene, in that a previously reported ENU germline mutant carrying a point mutation referred to as the NBT.
Mutagenesis in mice and rats using mobile DNA | Taconic Biosciences
Moreover, the liver phenotype in mice carrying the NBT. Hem1 is a member of the Hem family of cytoplasmic adaptor molecules predominantly and is expressed exclusively in hematopoietic cells, including T and B cells, macrophages, DCs and granulocytes. At the protein level, the inclusion of intronic nucleotides is predicted to result in a frame-shift and alternative coding following residue , and a premature stop at amino acid resulting in a largely truncated Hem-1 protein in Lampe2 mice Figure 3d.
The Human Genome Project has changed our approach to biology. Sequence data are being acquired from multiple organisms at a phenomenal pace and. The haploid size of the mouse genome is estimated to be ∼ Gb; hence even at the conservative mutation rate of one every Mb, each.
Given the similarities of the Lampe2 and NBT. Coarse mapping and identification of the causative mutation in Lampe2 mice.
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The Lampe2 phenotype was linked to the distal site of chromosome Hem1 is part of the Wiskott-Aldrich syndrome protein family Verprolin-homologous protein WAVE protein complex in hematopoietic cells regulating cell mobility and intracellular processes requiring rearrangement of the cytoskeleton following immuno-receptor activation, including B and T cell, chemokine and innate immune receptors such as Toll-like receptors.
Specifically, receptor triggering causes activation of Rho family of Guanosine triphosphatases GTPases such as CDC42, RhoA and Rac ultimately resulting in the activation of downstream adaptor complexes involved in the regulating of actin de polymerization. Under non-stimulated conditions, the WAVE complex is inactive, but following immunoreceptor activation, GTP-bound Rac binds the pentameric complex presumably through Sra1 In addition, this complex requires binding of phophatidylinositol 3,4,5 triphosphate PIP 3 interaction and phosphorylation by kinases 50 , including Abl kinase and Mitogen-activated protein kinases Interestingly, the absence of individual subunit components often causes the degradation of all components of the WAVE complex resulting in aberrant actin polymerization.
Assessing the immune system using ENU mutagenesis in mice has previously led to important breakthrough discoveries in understanding the genetics in human patients with PID. A prime example is the identification of the 3d allele—a missense allele of Unc93b1 , a gene encoding an ER membrane protein with 12 membrane spanning motifs with a previously unknown function. Homozygote 3d mutant mice were found to be unresponsive to ligands activating endosomal TLRs, but exhibited normal responses to TLRs expressed at the surface Interestingly, at the same time Casrouge et al.
Specifically, both patients were unresponsive to endosomal TLR stimulation and showed a high viral susceptibility.