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Mouse models of human cancers are important for understanding determinants of overt disease and for "preclinical" development of rational therapeutic strategies; for instance, based on macrodrugs. Chromosomal translocations underlie many human leukemias, sarcomas, and epithelial tumors. We have developed three technologies based on homologous recombination in mouse ES cells to mimic human chromosome translocations. The first, called the knockin method, allows creation of fusion genes like those typical of translocations of human leukemias and sarcomas. Two new conditional chromosomal translocation mimics have been developed. The first is a method for generating reciprocal chromosomal translocations de novo using Cre-loxP recombination (translocator mice). In some cases, there is incompatible gene orientation and the translocator model cannot be applied. We have developed a different model (invertor mice) for these situations. This method consists of introducing an inverted cDNA cassette into the intron of a target gene and bringing the cassette into the correct transcriptional orientation by Cre-loxP recombination. We describe experiments using the translocator model to generate MLL-mediated neoplasias and the invertor method to generate EWS-ERG-mediated cancer. These methods mimic the situation found in human chromosome translocations and provide the framework for design and study of human chromosomal translocations in mice.

Chromosomal translocations and other abnormalities are central to the initiation of cancer in all cell types. Understanding the mechanism is therefore important to evaluate the evolution of cancer from the cancer initiating events to overt disease. Recent work has concentrated on model systems to develop an understanding of the molecular mechanisms of translocations but naturally occurring events are more ideal case studies since biological selection is absent from model systems. In solid tumours, nonreciprocal translocations are most commonly found, and accordingly we have investigated the recurrent nonreciprocal t(3;5) chromosomal translocations in renal carcinoma to better understand the mechanism of these naturally occurring translocations in cancer. Unexpectedly, the junctions of these translocations can be associated with site-specific, intrachromosomal inversion involving at least two double strand breaks (DSB) in cis and rejoining by nonhomologous end joining or micro-homology end joining. However, these translocations are not necessarily associated with transcribed regions questioning accessibility per se in controlling these events. In addition, intrachromosomal deletions also occur. We conclude these naturally occurring, nonreciprocal t(3;5) chromosomal translocations occur after complex and multiple unresolved intrachromosomal DSBs leading to aberrant joining with concurrent interstitial inversion and that clonal selection of cells is the critical element in cancer development emerging from a plethora of DSBs that may not always be pathogenic. V C 2013 Wiley Periodicals, Inc.

Changes in DNA methylation patterns during embryo development and differentiation processes are linked to the transcriptional plasticity of our genome. However, little is known about the evolutionary conservation of DNA methylation patterns and the evolutionary impact of epigenetic differences between closely related species. Here we compared the methylation patterns of CpG islands (CGIs) in the promoter regions of seven genes in humans and chimpanzees. We identified a block of CpGs in the cell cycle-related kinase (CCRK) gene that is more methylated in the adult human cortex than in the chimpanzee cortex and, in addition, it exhibits considerable intraspecific variation both in humans and chimpanzees. The species-specifically methylated region (SMR) lies between the almost completely methylated 5' region and the completely demethylated 3' region of the presumed CCRK CGI promoter. It is part of an Alu-Sg1 repeat that has been integrated into the promoter region in a common ancestor of humans and New World monkeys. This SMR is relatively hypomethylated in the rhesus monkey cortex and more or less completely methylated in the baboon cortex, indicating extraordinary methylation dynamics during primate evolution. The mRNA expression level of CCRK has also changed during the course of primate evolution. CCRK is expressed at much higher levels in human and baboon cortices, which display an average SMR methylation of 70% and 100%, respectively, than in chimpanzee and rhesus macaque cortices with an average SMR methylation of 35% and 40%, respectively. The observed evolutionary dynamics suggests a possibility that CCRK has been important for evolution of the primate brain.

Human cancers and some congenital traits are characterized by cytogenetic aberrations including translocations, amplifications, duplications or deletions that can involve gain or loss of genetic material. We have developed a simple method to precisely delineate such regions with known or cryptic genomic alterations. Molecular copy-number counting (MCC) uses PCR to interrogate miniscule amounts of genomic DNA and allows progressive delineation of DNA content to within a few hundred base pairs of a genomic alteration. As an example, we have located the junctions of a recurrent nonreciprocal translocation between chromosomes 3 and 5 in human renal cell carcinoma, facilitating cloning of the breakpoint without recourse to genomic libraries. The analysis also revealed additional cryptic chromosomal changes close to the translocation junction. MCC is a fast and flexible method for characterizing a wide range of chromosomal aberrations. *Note: Correspondence should be addressed to M. Thangavelu (mt370@hutchison-mrc.cam.ac.uk) instead of T. H. Rabbitts. The error has been corrected in the PDF version of the article.

To study the genetics of atopy systematically we established a mouse model that provides the general phenotype of atopy: the early response characteristic of IgE-dependent eczema or atopic dermatitis, and the diagnostic test of atopy, the skin-prick test. Using an immediate cutaneous hypersensitivity test (ICHS) against birch pollen extract we could classify A/J and C57BL/6 (B6) inbred mouse strains respectively as high responder and low responders. The F1 hybrids were found to be high responders with incomplete penetrance. Backcrossing F1 mice to the low responder B6 strain yielded three classes of responders, high, intermediate, and low. A genome-wide microsatellite screen of the backcross progeny disclosed suggestive linkage to a microsatellite marker on chromosome 6 close to the locus of the IL-5 receptor alpha chain. Its allelic variation in A/J and B6 strains was investigated and two major differences were detected. Firstly, a nucleotide exchange in the 5' untranslated region of B6 mRNA resulted in increased transcription/translation of a reporter construct. Higher expression of the receptor on the cell surface would be expected to favor an allergic immune response. Secondly, the two alleles are differentially spliced so as to yield two soluble isoforms in A/J mice versus one in B6 mice. Higher expression of soluble IL-5R would be expected to reduce the level of allergy through capture of IL-5. Thus both findings conform to the expectation based on susceptibility to atopy and thus identify the IL-5R alpha chain as a likely contributor to the genetics of atopy.

For a series of immunological diseases including asthma, inflammatory arthritis and experimental allergic encephalomyelitis the non-classical major histocompatibility complex (MHC) genetics of man and mouse has been making rapid progress. Information is available not only for the disease associations of individual candidate genes but also from the first genome scans. In both species the proinflammatory cytokine genes and/or their related receptors and inhibitors (IL-1, IL-1r, IL-1ra, IL-2, IL-6r, TNF-alpha), and to a lesser extent the anti-inflammatory cytokine IL-4 are implicated as candidate control elements. In contrast, genes for the signalling and adhesion CD molecules have so far been inconspicuous. Most of the polymorphisms so far detected have been in the regulatory sequences of these genes, rather than in the exons. It is suggested that the benefit conferred on an individual by greater flexibility in its immunoregulatory machinery may be responsible for maintaining this form of polymorphism.

Reactive arthritis (ReA) is a T cell mediated inflammatory process. The immune response is primarily directed against a triggering organism, although autoimmunity has been invoked in long-lasting, antibiotic-resistant disease. Although a variety of different species are known to trigger Reactive arthritis, the clinical manifestations are strikingly similar as well as closely associated to the HLA-B27 (70%). Various antigenic fractions and single antigens of Yersinia enterocolitica were prepared, and their immunological activity was assessed by proliferation of synovial fluid mononuclear cells from 10 Reactive arthritis patients. The gene encoding one hitherto unknown antigen has been sequenced. Nonapeptides deduced from sequences of the target antigens were tested in an assembly assay. Two immunodominant proteins of Yersinia enterocolitica were found, one being the urease β-subunit and the other the 50 S ribosomal protein L23. The latter has been sequenced and belongs to the evolutionarily conserved ribosomal proteins with homology to procaryotes and eucaryotes. One nonapeptide derived from the urease β-subunit was identified as a possible epitope for HLA-B27-restricted cytotoxic T cells by its high affinity. This epitope is also highly conserved. Sharing of conserved immunodominant proteins between different disease triggering microorganisms could provide an explanation of the shared clinical picture in Reactive arthritis. Moreover, autoimmunity in Reactive arthritis might be mediated by antigen mimicry between evolutionarily conserved epitopes of ribosomal proteins and their host analogs.

A homozygous reciprocal translocation, 46,XY,t(10;11),t(10;11), was detected in a boy with non-syndromic congenital sensorineural hearing impairment. Both parents and their four other children were heterozygous translocation carriers, 46,XX,t(10;11) and 46,XY,t(10;11), respectively. Fluorescence in situ hybridization of region-specific clones to patient chromosomes was used to localize the breakpoints within bacterial artificial chromosome (BAC) RP11-108L7 on chromosome 10q24.3 and within BAC CTD-2527F12 on chromosome 11q23.3. Junction fragments were cloned by vector ligation and sequenced. The chromosome 10 breakpoint was identified within the PDZ domain containing 7 (PDZD7) gene, disrupting the open reading frame of transcript PDZD7-C (without PDZ domain) and the 5 0 -untranslated region of transcript PDZD7-D (with one PDZ and two prolin-rich domains). The chromosome 11 breakpoint was localized in an intergenic segment. Reverse transcriptase–polymerase chain reaction analysis revealed PDZD7 expression in the human inner ear. A murine Pdzd7 transcript that is most similar in structure to human PDZD7-D is known to be expressed in the adult inner ear and retina. PDZD7 shares sequence homology with the PDZ domain-containing genes, USH1C (harmonin) and DFNB31 (whirlin). Allelic mutations in harmonin and whirlin can cause both Usher syndrome (USH1C and USH2D, respectively) and congenital hearing impairment (DFNB18 and DFNB31, respectively). Protein–protein interaction assays revealed the integration of PDZD7 in the protein network related to the human Usher syndrome. Collectively, our data provide strong evidence that PDZD7 is a new autosomal-recessive deafness-causing gene and also a prime candidate gene for Usher syndrome.

A boy with severe mental retardation, funnel chest, bell-shaped thorax, and hexadactyly of both feet was found to have a balanced de novo t(12;17)(p13.3;q21.3) translocation. FISH with BAC clones and long-range PCR products assessed in the human genome sequence localized the breakpoint on chromosome 17q21.3 to a 21-kb segment that lies

MLL-AF4 fusion is the most common consequence of chromosomal translocations in infant leukaemia and is associated with a poor prognosis. MLL-AF4 is thought to be required in haematopoietic stem cells to elicit leukaemia and may be involved in tumour phenotype specification as it is only found in B-cell tumours in humans. We have employed the invertor conditional technology to create a model of MLL-AF4, in which a floxed AF4 cDNA was knocked into Mll in the opposite orientation for transcription. Cell-specific Cre expression was used to generate Mll-AF4 expression. The mice develop exclusively B-cell lineage neoplasias, whether the Cre gene was controlled by B- or T-cell promoters, but of a more mature phenotype than normally observed in childhood leukaemia. These findings show that the MLL-AF4 fusion protein does not have a mandatory role in multi-potent haematopoietic stem cells to cause cancer and indicates that MLL-AF4 has an instructive function in the phenotype of the tumour.

Chromosomal translocations are primary events in tumorigenesis. Those involving the mixed lineage leukaemia (MLL) gene are found in various guises and it is unclear whether MLL fusions can affect haematopoietic differentiation. We have used a model in which chromosomal translocations are generated in mice de novo by Cre-loxP-mediated recombination (translocator mice) to compare the functionally relevant haematopoietic cell contexts for Mll fusions, namely pluripotent stem cells, semicommitted progenitors or committed cells. Translocations between Mll and Enl or Af9 cause myeloid neoplasias, initiating in pluripotent stem cells or multipotent myeloid progenitors. However, while Mll-Enl translocations can also cause leukaemia from T-cell progenitors, no tumours arose with Mll-Af9 translocations in the T-cell compartment. Furthermore, Mll-Enl translocations in T-cell progenitors can cause lineage reassignment into myeloid tumours. Therefore, a permissive cellular environment is required for oncogenicity of Mll-associated translocations and Mll fusions can influence haematopoietic lineage commitment.

The marked association of abnormalities of chromosome 11 long arm, band q23, with human leukaemia led to the identification of the 11q23 gene called MLL (or HTRX, HRX, TRX1, ALL-1). MLL can become fused with one of a remarkable panoply of genes from other chromosome locations in individual leukaemias, leading to either acute myeloid or lymphoid tumours (hence the name MLL for mixed lineage leukaemia). The unusual finding that a single protein could be involved in both myeloid and lymphoid malignancies and that the truncated protein could do so as a fusion with very disparate partners has prompted studies to define the molecular role of MLL-fusions in leukaemogenesis and to the development of MLL-controlled mouse models of leukaemogenesis. These studies have defined MLL-fusion proteins as regulators of gene expression, controlling such elements as HOX genes, and have indicated a variety of mechanisms by which MLL-fusion proteins contribute to leukaemogenesis.

Chromosomal1 translocations are key elements in tumor etiology, as these somatically created abnormal chromosomes have activated oncogenes where the chromosome breakpoints occur (for review, see Rabbitts 1994). Most often, gene fusions are caused by chromosomal translocations, which frequently break within the exons of the two involved chromosomes, allowing the transcription product to encompass the linked exons, for post-transcriptional processing to splice these exons to create a tumor-specific fusion mRNA and in turn fusion protein. This type of event is common in both leukemias and in sarcomas (Rabbitts 1994; Look 1997). The leukemias divide into chronic and acute forms of cancer. The translocation genes involved can be roughly categorized into distinct types, with those involved in acute leukemia often being transcription regulators (Cleary 1991) whose role as master genes in cell fate determination is a key element in their role in leukemias (Rabbitts 1991). Thus, activated oncogenes or gene fusions influence differentiation in cell-specific ways and the tropism of specific chromosomal translocations for specific cell types is a manifestation of this role.

The etiology of human tumors often involves chromosomal translocations. Models that emulate translocations are essential to understanding the determinants of frank malignancy, those dictating the restriction of translocations to specific lineages, and as a basis for development of rational therapeutic methods. We demonstrate that developmentally regulated Cre-loxP-mediated interchromosomal recombination between the Mll gene, whose human counterpart is involved in a spectrum of leukemias, and the Enl gene creates reciprocal chromosomal translocations that cause myeloid tumors. There is a rapid onset and high penetrance of leukemogenesis in these translocator mice, and high proportions of cells carrying chromosomal translocations can be found in bone marrow as early as 12 days after birth. This de novo strategy is a direct recapitulation of naturally occurring human cancer-associated translocations.

Atopic disorders are complex traits with a significant contribution of heritability. Several mouse models for atopic phenotypes such as allergen-induced and intrinsic asthma, atopic dermatitis and immediate cutaneous hypersensitivity have been used to systematically dissect the genetics of these diseases. Many of the chromosomal regions identified in genome-wide screens colocalize with linkage regions of autoimmune phenotypes suggesting common regulatory genes in combination (or addition) to disease-specific genes. Moreover, many of these regions correspond to regions found in the human system, thus strengthening the likelihood of a gene contributing to the phenotype. The role of individual candidate genes has been vigorously pursued in genetically modified mice by destruction or overexpression of the candidate gene. The complex interactions of multiple contributing genes may further be elucidated in randomly mutated mouse strains. Thus mouse models can contribute largely to the elucidation of disease genes.

Asthma is a disease characterized by intermittent airway obstruction, inflammatory cell infiltrates, increased mucus production, lung epithelial remodeling, and airway hyperreactivity. The genetics of asthma, as investigated in animal models, is poorly understood. Because no animal model of asthma mimics all of the pathologic and physiological features of asthma, genetic studies have focused on several phenotypes, including intrinsic or native airway hyperreactivity. It is generally accepted that both genetic and environmental factors determine the phenotypic expression of this complex disease. The genetics of airway hyperresponsiveness, as investigated in the mouse, are presented in this review. The inbred mouse currently represents the most valuable genetic resource for understanding the factors that control this complex phenotype.

To facilitate whole-genome scan experiments, we selected a panel of 128 microsatellite markers on the basis of spacing and polymorphism in the strains DBA/2, BALB/c, AKR, C57BL/6, C57BL/10, A/J, C3H, 129/J, SJL/J, JF1, and PWB. Many of the primer pairs were redesigned for better performance. The last four strains were not characterized previously using these markers. JF1 and PWB are particularly interesting for intersubspecific crosses offering high polymorphism. We provide allele size data for the markers on these strains and add them to the emerging radiation hybrid framework map, which is not continuous except for chromosome 17 and 13. Information on the interrelationships of strains is useful both because of the importance of polymorphism in designing crosses and the background in assessing phenotypes. Microsatellites offer a widely dispersed, selectively neutral set of characters that lends itself conceptually to parsimony methods of analysis. The microsatellite allele size data were recoded as binary discrete characters in such a way that adjacent sizes differ by one step. Trees were generated using a Wagner parsimony method. As expected, the non-Mus domesticus strains, PWB (musculus) and JF1 (molossinus), are excluded from the domesticus strains. Among the domesticus strains, C57BL/6 and C57BL/10 (derived from the same founding pair) form a strongly supported group, as do C3H, A/J, and BALB/c (derived from the Bagg albino stock). No unique branching order for SJL/J, AKR, and DBA/2 is strongly supported, which may reflect a complicated history. Strain 129/J is clearly placed as the most deeply diverged of thedomesticus strains represented.

Genetic predisposition and environmental factors modulate the expression of allergic phenotypes. The frequent allergic phenotype 'immediate cutaneous hypersensitivity' was established in mice as a model for atopy. Genetic dissection of this trait requires a robust procedure to assess the allergic phenotype. To this end, different mouse strains were immunized with birch pollen extract. Immediate cutaneous hypersensitivity reactions were induced through intradermal allergen exposure. Wheel formation was quantitated and expressed as a hypersensitivity score according to the bonitur method. This procedure identified A/J and C57BL/6 mice as high– and low–responder strains, respectively. Crosses of A/J and C57BL/6 mice should allow the characterization of mendelian factors responsible for the two extreme phenotypes identified here.

Publisher Summary Genetic variation in humans and other diploid organisms results mainly from the presence in the population of polymorphic genes. A useful distinction, made possible by molecular genetics, is between variation in structural genes and variation in noncoding, regulatory regions. Regulatory regions are generally upstream of the corresponding structural genes, but may include introns and downstream segments. Within the immune system, polymorphism of structural segments is conspicuous only among genes encoding the antigen-handling proteins. The best known of these are the immunoglobulins, the T-cell receptors, and the class I and II major histocompatibility complex (MHC) proteins. Polymorphisms of the two proinflammatory cytokines IL-1 and tumor necrosis factor α (TNFα) are repeatedly associated with immunological diseases, which also apply to the antagonist IL-1RA and probably also to the receptor IL-1R. The association of these cytokine polymorphisms with immunological diseases occurs so regularly that they may well become a defining characteristic of these diseases. Polymorphism in upstream regulatory regions of MHC genes is conspicuous in class II MHC genes of both humans and mice. It may provide differential expression of these molecules in diverse antigen-presenting cells, and may thus influence the balance of Th1 and Th2 cytokines.