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.
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.
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.
Given the similarities of the Lampe2 and NBT. Coarse mapping and identification of the causative mutation in Lampe2 mice. 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. Following the identification of the causative mutation in 3d mice as being a missense allele of Unc93b1 , subsequent sequencing of the human patients indeed revealed aberrant mutations in UNC93B This example highlights the power of ENU mutagenesis and its unbiased approach, by uncovering the function of genes for which a biological function is otherwise difficult to predict.
With regard to the Gimap5 and Hem-1 germline mutations described in this chapter, both present examples of genetic mutations leading to severe combined immunodeficiencies. Although limited information is available with regard to genetic mutations causing a null phenotype in human GIMAP5 or HEM1, ample evidence exist that dysregulation of these genes plays an important role in human disease. Finally, perhaps due to its indispensable role in a wide variety of immune pathways, mutations in human HEM1 leading to dysregulated actin polymerization, have thus far not been reported.
Nonetheless, over- or under-expression of HEM1 is associated with disease prognosis in leukemia Following the breeding of the ENU-treated males to females homozygous for a dominant coat colour marker, the G1 founders are mated to mice carrying the specific chromosomal deletion and marked with a different coat colour marker. Four different classes of offspring are produced that can be distinguished by coat colour, from which it can be determined which will be useful Figure 2 Rinchik et al.
The resultant G2 mice can be screened for infertility or any other phenotype. Breeding strategies using deletions and inversions to generate mice homozygous for chromosome—specific ENU-induced mutations. The G1 founders are then mated to mice carrying a deletion marked with a different coat colour marker. Two classes of the resulting G2 offspring identified by coat colour will be useful, the test class that can be screened for novel phenotypes and the carrier class. The other two classes will be uninformative Brown and Balling, This inversion is also marked with a coat colour marker and is lethal in the homozygous state.
The G1 founders are then crossed with mice that are heterozygous for the balancer and another coat colour marker making it possible to distinguish all classes of progeny at the G2 stage. Mice carrying the balancer and the mutagenized chromosome are intercrossed to generate three classes of progeny that are identified by coat colour. The test class can be screened for novel phenotypes Brown and Balling, ; Kile et al. A similar but alternative approach is the use of mice with chromosome inversions in which mice with a balanced inversion are used.
The chromosome is also marked with a coat colour marker and is lethal in the homozygous state. The ENU-treated male is mated to mice carrying an inversion.
The G1 founders are then crossed with mice that are heterozygous for the balancer and a different coat marker that allows selection of mice at the G2 stage. Only mice known to be carrying the balancer and the mutagenized are inter-crossed to produce three classes of G2 mice that are readily distinguishable based on coat type Figure 2 B.
An ENU screen using a 24cM inversion on chromosome 11 has identified, among other abnormalities, three independent mutations affecting male fertility Kile et al. In addition to the 88 genes that this group mapped to the inverted region on chromosome 11, a further recessive mutant phenotypes were found that were not linked to the inverted region Pask et al. One of the non-linked mutations was subsequently identified as being within the GnRH receptor gene and resulted in hypogonadotrophic hypogonadism Pask et al.
For screens using balancer chromosomes or chromosome deletions, only mutations occurring within the inverted or deleted regions can be rapidly defined. These strategies offer the advantage that the mutation is immediately mapped to a relatively small pre-defined region of the genome.
Specific mutations are usually identified by the sequencing of candidate genes from within the region. This strategy is currently limited by the poor, but improving, availability of chromosomal deletions Russ et al. Once a phenotype of interest had been identified the next step is to localize the causal mutation. To do this, a known carrier of the mutation needs to be outbred to a wild-type mouse of a different genetically inbred strain; e. With each generation of subsequent breeding, the chromosomes become more and more of a mix of the two strains e.
All outbred mice displaying the abnormal phenotype will carry the causal mutation embedded within a region of chromosome derived from the original mutant strain i. The region carrying the causal mutation can be identified using a number of mapping techniques see below and Figure 3.
A large number of mice are, however, usually required to map the mutation to a manageable chromosomal region e. It is of particular note that as the phenotype of interest is infertility, homozygous males cannot be used for breeding. This involves breeding the ENU-treated male to a wild-type mouse of a different mouse strain rather than a wild-type mouse of the same strain.
For male infertility phenotypes, this should save two generations of breeding compared to the strategy described in Figure 1. This approach can, however, be complicated by modifier genes in the second strain leading to phenotype variation.
Microsatellites are simple, tandemly repeated di- to tetra-nucleotide sequence motifs flanked by unique sequences. They are valuable as genetic markers, because they are easily and economically assayed by the PCR McCouch et al. By selecting markers that differ in length between two mouse strains, a simple PCR amplification can be done to identify the strain origin of a particular chromosome region within a mixed background mouse Figure 3 B.
Markers that differ between the two mouse strains e. Outbreeding and mapping strategies to identify a chromosomal region containing an ENU-induced mutation. To identify the chromosomal region in which the ENU-induced mutation resides, there are several methods. Following scanning of the entire genome, a bias to the founder strain e. Further fine mapping within this region followed by selection of candidate genes will identify the causal ENU-mutated gene. By scanning the entire autosome using PCR amplification of selected microsatellites that differ in length between the two mouse strains used and comparing mice displaying the abnormal phenotype to unaffected sibling controls, a bias should be seen towards chromosomal regions of the original mouse strain e.
The mutation will be contained within one of the regions showing a strong bias to the original mutation strain. By identifying a large chromosomal region initially, then refining the linkage region using more closely spaced markers followed by the sequencing of candidate genes, the causal gene can be identified. Similar to mapping using microsatellite markers, single-nucleotide polymorphisms SNPs can be used to identify the chromosomal region in which the ENU-induced mutation resides.
SNPs are a sequence polymorphism that differs in a single base pair between mouse strains or individuals. By analysing SNPs distributed throughout the entire genome that differ between the original mutated mouse strain and the outbreding mouse strain, as with the above technique, a chromosomal region can be identified that shows a strong bias to SNPs from the original mutated mouse strain. Additional SNP mapping to further narrow down this region can then be done using more closely spaced polymorphisms, followed by the sequencing of candidate genes.
SNP genotyping has the advantage over microsatellite markers as there are usually many more SNP variations between strains than there are microsatellite length differences. Their disadvantage is however, that their analysis usually requires expensive and specialized equipment, as opposed to microsatellite markers that require only PCR. These two techniques are often used in combination, beginning with microsatellite mapping to crudely link the chromosomal region then further narrowing of the region using SNPs.
As outlined in Table I , there are many effects a single point mutation caused by ENU can have on the gene product. To determine the function of a particular gene, G1 or G2 mouse DNA can be screened for mutations within a gene of interest using a range of mutation detection techniques including denaturing DHPLC, direct sequencing and chemical cleavage.
With the high mutation load in the G1 mice, this approach is feasible and may produce mice with phenotypes more reflective of human disease than traditional knockouts i. Several different mutations within the same gene should be found if a sufficiently large number of G1 mice can be obtained.
For example, assuming there are 30 functional mutations within a G1 mouse and there are approximately 30 genes within the genome Claverie, , screening mice should on average identify one mutation in every gene. As previously mentioned, an allelic series of mutations is extremely valuable in understanding the function of a gene. Seven new ENU-induced mutations were identified and analysed in combination with two previously known mutant lines.
Five of the lines carried mis-sense mutations, one carried a nonsense mutation and resulted in exon skipping and the other affected a splice site. Each of these mutations provided important information on the structural requirements for the function of KitL and its function in several processes, including male fertility Rajaraman et al. Both Ingenium Pharmaceuticals Augustin et al.
They have generated large numbers of ENU-mutant G1 mice and archived both sperm and DNA to enable the rapid identification and regeneration of mouse lines of interest. The RIKEN group have screened G1 mice for 63 target loci and have identified ENU-induced mutations within this region using temperature gradient capillary electrophoresis followed by direct sequencing Sakuraba et al.
Large archives such as these two provide a complementary and cost-competitive alternative to gene-knockout technologies and hold the potential to generate allelic series for the dissection of gene function. Although ENU mutagenesis programs worldwide have been effective in identifying interesting mutants followed by the successful mapping of the causal gene, it must be noted that this process requires large colony numbers and personnel.
Additionally, the identification of these mutated genes can be somewhat challenging. These large-scale screens are capable of producing many mutant mouse lines, of which it would not be possible for interested investigators to follow up on every single one at the time of identification. This highlights the importance of an efficient and effective sperm archiving system by which mouse lines of interest can be generated at a later date.
Such archiving will ensure that these identified mutants will produce many results in the years to come. Additionally, researchers must be encouraged to use established programs. This will not only increase publication output but will drive down the per mouse costs for the whole project. The majority of infertile men demonstrate a defect in sperm production and, for most, the cause of their abnormal semen patterns are unknown idiopathic infertility de Kretser and Baker, Increasingly, it is recognized that the aetiology of the infertility will be genetic McLachlan et al.
In addition to the knowledge that can be gained from the identification of new genes involved in male fertility, identification of new genes that cause infertility when mutant may aid in the development and treatment of male infertility and also the development of new male-based contraceptives.
Studies into the mechanisms of spermatogenesis have uncovered many genetic determinants for male infertility. Disruptions at any of these stages can result in infertility. With the exception of vasectomy and condoms, there are currently no male-based contraceptives that are widely available. Of the different experimental methods of male contraception, a hormonal approach is the closest to being implemented with the pharmaceutical companies, Schering and Organon, recently beginning clinical trials using testosterone and progestin which temporarily suppress the hypothalamus—hypophyseal axis that stimulates sperm production Mandavilli, As a consequence of the suppression of testicular steroidogenesis, hormonal approaches can have a variety of side effects, [reviewed in Lyttle and Kopf, ].
An immunological approach has also been employed in the development of male-based contraceptives using sperm-specific antigens which lead to the development of circulating or reproductive tract anti-sperm antibodies.
The criteria for selection of a suitable antigen are important and include the localization of the antigen on the sperm surface or oocyte , and the ability of bound antibodies to interfere with function e.
The efficiency of this immunological approach in humans has been varied Moudgal et al. There have been a wide range of undesirable side effects reported and efficacy appears to be significantly influenced by racial origin.
This approach is now widely considered as unsuitable for humans Primakoff et al. Most recently, researchers have focused on identifying targets for non-hormonal contraception so as to take advantage of the cellular and physiological processes unique to the reproductive organs [reviewed in Lyttle and Kopf, ]. The main goal of this approach is to interfere in a highly specific manner in key processes involved in spermatogenesis, epididymal sperm maturation or sperm function.
This approach requires the identification of many unknown genes. Theoretically at least, this goal is achievable because of the high number of male reproductive tract-specific genes or isoforms of somatic genes. Clinically, we are yet to see the outcomes of this approach.
ENU mutagenesis has already been proven by many laboratories as an efficient and successful way to produce mice displaying any phenotype of interest, including abnormal male fertility.
Additionally, with advances being made in mapping techniques and the continued identification of markers particularly SNPs , linking the causal gene will only get easier. As a result of the identification of new genes and mutations using ENU-induced mouse models, significant advances can be made in the diagnosis and treatment of infertile men and the development of new male gamete-based contraceptives.
Endocr Rev 23 , — Mamm Genome 16 , — Balling R ENU mutagenesis: analyzing gene function in mice. Annu Rev Genomics Hum Genet 2 , — Beier DR Sequence-based analysis of mutagenized mice. Mamm Genome 11 , — Curr Opin Genet Dev 11 , — Clark AT , Firozi K and Justice MJ Mutations in a novel locus on mouse chromosome 11 resulting in male infertility associated with defects in microtubule assembly and sperm tail function. Biol Reprod 70 , — Claverie JM Gene number.
What if there are only 30, human genes? Science , — Semin Cell Dev Biol 9 , — Nat Genet 25 , — ENU mutagenesis to isolate mutations. These lesions are present in the sperm of the male, and after their isolation through genetic and phenotypic screens, give rise to a variety of phenotypic mutations. The mutant protein products are primarily missense mutations, a valuable class of mutations for dissecting protein structure and function.
Mutagenesis in the mouse will emphasize modeling human diseases through pheno-type-driven assays. Different genetic screens can be used to isolate ENU-induced mutations. A single generation screen can rapidly generate viable and fertile mutants that represent allele series, modifiers or dominant mutations. Two-generation deletion screens can identify recessive lethal mutations in a defined region of the genome. Three-generation pedigree screens may be used to scan the entire genome for a viable mutation of interest or, in combination with linked markers or balancer chromosomes, to isolate lethal or sterile alleles see below.
Several large-scale mutagenesis screens have already been funded internationally. The key features ofthese screens are summarized in Table 3. Each ofthese screens uses a different genetic strategy to isolate mutations: some screens target dominant mutations, whereas others are designed to isolate recessive mutations.
Some groups are screening for recessive lethal and detrimental mutations with regional mutagenesis to address gene function in parallel with the Human Genome Project. Other groups are scanning large numbers of mutagenized genomes for dominant neurological and clinical hematology or biochemical variants. Screening for mutations does not have to be carried out on a large scale. Two cost-effective strategies for the small laboratory are allelic series and sensitized pathway screens.
A sensitized pathway screen targets a single biological or biochemical pathway, and exploits non-allelic non-complementation to isolate mutations in the first generation offspring of mutagenized males. Some sensitized screens may be carried out in the background of a drug or environmental modification, instead of a genetic modification. For example, using a phenylalanine injection as a sensitizer, a number of mutations affecting the phenylalanine metabolism pathway were isolated as heterozygotes 20 , A unique approach towards isolating mutations combines genebased targeting in embryonic stem cells with phenotype-driven ENU mutagenesis.
Powerful genetic strategies in Drosophila rely on the availability of genetic reagents such as deletions, duplications and inversions to facilitate genetic screens and provide simple, cost-effective stock maintenance. Deletions are useful for generating haploidy in genetic screens, as well as for mapping using non-complementation strategies Fig. Inversions that carry a dominant marker and are homozygous lethal are ideal balancer chromosomes to suppress recombination and readily identify classes of offspring for the isolation and maintenance of lethal or detrimental mutations Fig.
The approach is gene-based because the endpoints of the deletion or inversion are known, minimizing the amount of effort required to characterize the rearrangements. Engineering techniques allow for the rearrangements to be tagged with a dominant yellow coat color marker, K agouti , providing a resource for simple mapping, stock maintenance and genetic screens 22 , 24 , The creation of mouse genomic libraries containing the constructs necessary for the targeting events provides a rapid system for their generation anywhere in the mouse genome Fig.
An initial mutagenesis effort, designed to isolate recessive mutations of many phenotypic classes, is targeting mouse Chromosome 11, which is a gene rich chromosome highly conserved with human Chromosome The goal is to saturate the chromosome with mutations to define gene function, then use linkage conservation between the mouse and human to predict gene function in the human. Both schemes take advantage of the yellow coat color conferred by the K14 -agouti transgene.
The deletion scheme can be used only for large deletions that are not detrimental to the animal, limiting the screen to certain regions, but allowing mutations to be isolated in only two generations. The inversion scheme allows a larger portion of the chromosome to be screened for mutations, but requires three generations of breeding. To be valuable, new mutations must be localized so that candidate genes and relevant human disease models can be identified. Point mutations isolated by their phenotype must be mapped using phenotype information, since a molecular tag is not available.
Traditionally, these mutations are mapped in meiotic backcrosses segregating the phenotype relative to multiple molecular polymorphisms for a review see ref. To map to a resolution of 10 cM requires analyzing meioses.
Thus, new mutations isolated genome-wide would require the analysis of 10 mice. High resolution mapping strategies for positional cloning usually involve the analysis of — meioses per mutation.
A multitude of dominant and recessive mutations can be isolated in any ENU screen, making mapping the bottleneck in the process, and requiring simpler mapping technologies such as DNA pooling The coat color tagged screens described above are unique in that the mutation is isolated linked to visible chromosomal markers, so its chromosome location is known upon isolation, eliminating the need for meiotic mapping.
Because the sequence of the mouse genome will soon be available, many mutations will be assigned to genes based on positional candidacy after their localization. In addition, BAC complementation can be used to identify mutation-gene correlations Two lambda mouse genomic libraries have been constructed that contain the selectable markers required for two step targeting events. If the loxP sites are inserted in cis in the same orientation, recombination after Cre transfection will produce a deletion, and HAT resistant, Puro sensitive, Neo sensitive ES cells.
If the loxP sites are inserted in opposite orientation, recombination after Cre transfection will result in an inversion, with HAT resistant, Puro resistant, Neo resistant ES cells. B and C Mutagenesis schemes for mouse Chromosome 11 using yellow coat color-tagged chromosomal rearrangements. The deletion or inversion is tagged with a dominant yellow coat color marker, Kagouti yellow. Each scheme uses the dominant Rex Re , and indicated by the black chromosome mutation on Chr 11, which causes curly fur mottled.
B The deletion scheme. The Re mutation marks the non-mutagenized chromosome, with the caveat that recombination can occur between a new linked mutation and Re.
G1 animals, heterozygous for ENU mutagenized chromosomes and Re are mated to mice hemizygous for a yellow-tagged deletion. The resulting classes ofoffspring can be readily identified: i the mutant class is yellow and straight-haired and, if missing, indicates the likelihood of a lethal mutation; ii a carrier class that is wild-type, and canbeusedto recover any lethal mutations; iii two curly-haired classes of mice black and yellow that are uninformative and can be immediately discarded.
C The inversion scheme. The balancer chromosome contains an inversion that suppresses recombination over a reasonable interval, cM, is marked with the dominant Kagouti transgene conferring yellow coat color, and is homozygous lethal due to disruption of one or more lethal genes at its endpoints.
After regaining fertility, ENU-treated males are mated to females carrying the balancer chromosome yellow. G1 animals that are yellow are mated with animals heterozygous for the balancer chromosome and Re yellow mottled. Three classes of offspring can be identified in the second generation, and the fourth class, which is homozygous for the balancer chromosome, dies upside down. The useful G2 animals are the yellow, straight-haired animals, which are brother-sister mated. The G3 offspring are easily classified as: i the wild-type mutant class, which if missing indicates the likelihood of a linked lethal mutation and ii a carrier class usedtorescueany lethal mutations, which carries the balanced point mutation, ideal for stock maintenance.
As new technologies are being developed for high-throughput single nucleotide polymorphism detection, technologies for the detection of point mutations will become simpler 30 , Unlike humans, naturally occurring polymorphisms are rare in inbred strains of mice, in particular within coding regions.
Thus, the detection of point mutations is possible in an inbred strain background, making mutation detection with mismatch repair enzymes a potential approach to quickly map and identify the lesions. In any screen for mutations using ENU, mutation isolation relies upon the phenotype assay, requiring that the mutant phenotype must vary significantly from the background. However, screening for mutations involves the analysis ofmany animals, so phenotype screens must be broad and inexpensive.
Visible phenotypes that affect the eye, coat, size or neurological behavior are simple to identify, and such screens often yield novel mutations. Behavior and sensory organ phenotypes can be screened for using standard tests for reflex, sight or hearing loss, motor development, balance and coordination, as well as learning and memory.
Skeletal development and soft tissue morphology can be examined using high resolution, low energy X-ray analysis. Clinical tests performed on mouse blood can yield a vast array of phenotypes relevant to human clinical disease even though tests performed on mouse body fluids must be performed on a microscale.
Because of existing microscale tests for human infants, many clinical tests are already available. A complete blood count with microscopic differential analysis can identify abnormalities in red blood cell and white blood cell numbers or morphology, as well as platelet abnormalities. Extending the analysis of blood cells with flow cytometry may uncover other immunological defects.
Antinuclear antibody quantitation can detect serum autoantibodies in a large variety of autoimmune disorders, including systemic lupus erythematosus.
Clinical chemistry tests can diagnose multiple organ system anomalies, including liver, pancreatic, heart and kidney disorders. Urinalysis on mice reveals increased levels of protein or other abnormal by-products of disease. Tandem mass spectrometry can detect a variety of metabolic disorders affecting lipids, fatty acids or amino acids. Additional assays are being developed to identify neurological, cardiovascular, hematopoietic and immune phenotypes using high-throughput technologies such as microarrays.
A mutant mouse resource.
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