Publications by authors named "Ramiro Ramirez-Solis"

48 Publications

High-throughput phenotyping reveals expansive genetic and structural underpinnings of immune variation.

Nat Immunol 2020 01 16;21(1):86-100. Epub 2019 Dec 16.

Wellcome Sanger Institute, Hinxton, UK.

By developing a high-density murine immunophenotyping platform compatible with high-throughput genetic screening, we have established profound contributions of genetics and structure to immune variation (http://www.immunophenotype.org). Specifically, high-throughput phenotyping of 530 unique mouse gene knockouts identified 140 monogenic 'hits', of which most had no previous immunologic association. Furthermore, hits were collectively enriched in genes for which humans show poor tolerance to loss of function. The immunophenotyping platform also exposed dense correlation networks linking immune parameters with each other and with specific physiologic traits. Such linkages limit freedom of movement for individual immune parameters, thereby imposing genetically regulated 'immunologic structures', the integrity of which was associated with immunocompetence. Hence, we provide an expanded genetic resource and structural perspective for understanding and monitoring immune variation in health and disease.
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http://dx.doi.org/10.1038/s41590-019-0549-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7338221PMC
January 2020

Reduced lifespan of mice lacking catalase correlates with altered lipid metabolism without oxidative damage or premature aging.

Free Radic Biol Med 2019 05 25;135:102-115. Epub 2019 Feb 25.

Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mor, Mexico. Electronic address:

The relationship between the mechanisms that underlie longevity and aging and the metabolic alterations due to feeding conditions has not been completely defined. In the present work, through the deletion of the gene encoding catalase, hydrogen peroxide (HO) was uncovered as a relevant regulator of longevity and of liver metabolism. Mice lacking catalase (Cat) fed ad libitum with a regular diet showed a shorter lifespan than wild type mice, which correlated with reduced body weight, blood glucose levels and liver fat accumulation, but not with increased oxidative damage or consistent premature aging. High fat diet (HFD) and fasting increased oxidative damage in the liver of wild type animals but, unexpectedly, this was not the case for that of Cat mice. Interestingly, although HFD feeding similarly increased the body weight of Cat and wild-type mice, hyperglycemia and liver steatosis did not develop in the former. Fat accumulation due to fasting, on the other hand, was diminished in mice lacking catalase, which correlated with increased risk of death and low ketone body blood levels. Alteration in expression of some metabolic genes in livers of catalase deficient mice was consistent with reduced lipogenesis. Specifically, Pparγ2 expression up-regulation in response to a HFD and down-regulation upon fasting was lower and higher, respectively, in livers of Cat than of wild type mice, and a marked decay was observed during Cat mice aging. We propose that catalase regulates lipid metabolism in the liver by an evolutionary conserved mechanism that is determinant of lifespan without affecting general oxidative damage.
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http://dx.doi.org/10.1016/j.freeradbiomed.2019.02.016DOI Listing
May 2019

Genome editing: A perspective on the application of CRISPR/Cas9 to study human diseases (Review).

Int J Mol Med 2019 Apr 26;43(4):1559-1574. Epub 2019 Feb 26.

Universidad Autónoma de Nuevo León, Department of Biochemistry and Molecular Medicine, School of Medicine and University Hospital 'Dr. José E. González', Monterrey, Nuevo León 64460, México.

Genome editing reemerged in 2012 with the development of CRISPR/Cas9 technology, which is a genetic manipulation tool derived from the defense system of certain bacteria against viruses and plasmids. This method is easy to apply and has been used in a wide variety of experimental models, including cell lines, laboratory animals, plants, and even in human clinical trials. The CRISPR/Cas9 system consists of directing the Cas9 nuclease to create a site‑directed double‑strand DNA break using a small RNA molecule as a guide. A process that allows a permanent modification of the genomic target sequence can repair the damage caused to DNA. In the present study, the basic principles of the CRISPR/Cas9 system are reviewed, as well as the strategies and modifications of the enzyme Cas9 to eliminate the off‑target cuts, and the different applications of CRISPR/Cas9 as a system for visualization and gene expression activation or suppression. In addition, the review emphasizes on the potential application of this system in the treatment of different diseases, such as pulmonary, gastrointestinal, hematologic, immune system, viral, autoimmune and inflammatory diseases, and cancer.
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http://dx.doi.org/10.3892/ijmm.2019.4112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6414166PMC
April 2019

Identification of genes required for eye development by high-throughput screening of mouse knockouts.

Commun Biol 2018 21;1:236. Epub 2018 Dec 21.

Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, 77030, USA.

Despite advances in next generation sequencing technologies, determining the genetic basis of ocular disease remains a major challenge due to the limited access and prohibitive cost of human forward genetics. Thus, less than 4,000 genes currently have available phenotype information for any organ system. Here we report the ophthalmic findings from the International Mouse Phenotyping Consortium, a large-scale functional genetic screen with the goal of generating and phenotyping a null mutant for every mouse gene. Of 4364 genes evaluated, 347 were identified to influence ocular phenotypes, 75% of which are entirely novel in ocular pathology. This discovery greatly increases the current number of genes known to contribute to ophthalmic disease, and it is likely that many of the genes will subsequently prove to be important in human ocular development and disease.
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http://dx.doi.org/10.1038/s42003-018-0226-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303268PMC
December 2018

A synthesis approach of mouse studies to identify genes and proteins in arterial thrombosis and bleeding.

Blood 2018 12 1;132(24):e35-e46. Epub 2018 Oct 1.

Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands.

Antithrombotic therapies reduce cardiovascular diseases by preventing arterial thrombosis and thromboembolism, but at expense of increased bleeding risks. Arterial thrombosis studies using genetically modified mice have been invaluable for identification of new molecular targets. Because of low sample sizes and heterogeneity in approaches or methodologies, a formal meta-analysis to compare studies of mice with single-gene defects encountered major limitations. To overcome these, we developed a novel synthesis approach to quantitatively scale 1514 published studies of arterial thrombus formation (in vivo and in vitro), thromboembolism, and tail-bleeding of genetically modified mice. Using a newly defined consistency parameter (CP), indicating the strength of published data, comparisons were made of 431 mouse genes, of which 17 consistently contributed to thrombus formation without affecting hemostasis. Ranking analysis indicated high correlations between collagen-dependent thrombosis models in vivo (FeCl injury or ligation/compression) and in vitro. Integration of scores and CP values resulted in a network of protein interactions in thrombosis and hemostasis (PITH), which was combined with databases of genetically linked human bleeding and thrombotic disorders. The network contained 2946 nodes linked to modifying genes of thrombus formation, mostly with expression in megakaryocytes. Reactome pathway analysis and network characteristics revealed multiple novel genes with potential contribution to thrombosis/hemostasis. Studies with additional knockout mice revealed that 4 of 8 (, , , ) new genes were modifying in thrombus formation. The PITH network further: (i) revealed a high similarity of murine and human hemostatic and thrombotic processes and (ii) identified multiple new candidate proteins regulating these processes.
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http://dx.doi.org/10.1182/blood-2018-02-831982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6293874PMC
December 2018

Establishment of mouse expanded potential stem cells.

Nature 2017 10 11;550(7676):393-397. Epub 2017 Oct 11.

Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan.

Mouse embryonic stem cells derived from the epiblast contribute to the somatic lineages and the germline but are excluded from the extra-embryonic tissues that are derived from the trophectoderm and the primitive endoderm upon reintroduction to the blastocyst. Here we report that cultures of expanded potential stem cells can be established from individual eight-cell blastomeres, and by direct conversion of mouse embryonic stem cells and induced pluripotent stem cells. Remarkably, a single expanded potential stem cell can contribute both to the embryo proper and to the trophectoderm lineages in a chimaera assay. Bona fide trophoblast stem cell lines and extra-embryonic endoderm stem cells can be directly derived from expanded potential stem cells in vitro. Molecular analyses of the epigenome and single-cell transcriptome reveal enrichment for blastomere-specific signature and a dynamic DNA methylome in expanded potential stem cells. The generation of mouse expanded potential stem cells highlights the feasibility of establishing expanded potential stem cells for other mammalian species.
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http://dx.doi.org/10.1038/nature24052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5890884PMC
October 2017

Prevalence of sexual dimorphism in mammalian phenotypic traits.

Nat Commun 2017 06 26;8:15475. Epub 2017 Jun 26.

Mouse Genetics Project, The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.

The role of sex in biomedical studies has often been overlooked, despite evidence of sexually dimorphic effects in some biological studies. Here, we used high-throughput phenotype data from 14,250 wildtype and 40,192 mutant mice (representing 2,186 knockout lines), analysed for up to 234 traits, and found a large proportion of mammalian traits both in wildtype and mutants are influenced by sex. This result has implications for interpreting disease phenotypes in animal models and humans.
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http://dx.doi.org/10.1038/ncomms15475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5490203PMC
June 2017

TCTE1 is a conserved component of the dynein regulatory complex and is required for motility and metabolism in mouse spermatozoa.

Proc Natl Acad Sci U S A 2017 07 19;114(27):E5370-E5378. Epub 2017 Jun 19.

State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 210029, People's Republic of China;

Flagella and cilia are critical cellular organelles that provide a means for cells to sense and progress through their environment. The central component of flagella and cilia is the axoneme, which comprises the "9+2" microtubule arrangement, dynein arms, radial spokes, and the nexin-dynein regulatory complex (-DRC). Failure to properly assemble components of the axoneme leads to defective flagella and in humans leads to a collection of diseases referred to as ciliopathies. Ciliopathies can manifest as severe syndromic diseases that affect lung and kidney function, central nervous system development, bone formation, visceral organ organization, and reproduction. T-Complex-Associated-Testis-Expressed 1 (TCTE1) is an evolutionarily conserved axonemal protein present from (DRC5) to mammals that localizes to the -DRC. Here, we show that mouse TCTE1 is testis-enriched in its expression, with its mRNA appearing in early round spermatids and protein localized to the flagellum. TCTE1 is 498 aa in length with a leucine rich repeat domain at the C terminus and is present in eukaryotes containing a flagellum. Knockout of results in male sterility because -null spermatozoa show aberrant motility. Although the axoneme is structurally normal in mutant spermatozoa, -null sperm demonstrate a significant decrease of ATP, which is used by dynein motors to generate the bending force of the flagellum. These data provide a link to defining the molecular intricacies required for axoneme function, sperm motility, and male fertility.
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http://dx.doi.org/10.1073/pnas.1621279114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5502601PMC
July 2017

A staging system for correct phenotype interpretation of mouse embryos harvested on embryonic day 14 (E14.5).

J Anat 2017 May 9;230(5):710-719. Epub 2017 Feb 9.

Centre for Anatomy and Cell Biology & MIC, Medical University of Vienna, Vienna, Austria.

We present a simple and quick system for accurately scoring the developmental progress of mouse embryos harvested on embryonic day 14 (E14.5). Based solely on the external appearance of the maturing forelimb, we provide a convenient way to distinguish six developmental sub-stages. Using a variety of objective morphometric data obtained from the commonly used C57BL/6N mouse strain, we show that these stages correlate precisely with the growth of the entire embryo and its organs. Applying the new staging system to phenotype analyses of E14.5 embryos of 58 embryonic lethal null mutant lines from the DMDD research programme (https://dmdd.org.uk) and its pilot, we show that homozygous mutant embryos are frequently delayed in development. To demonstrate the importance of our staging system for correct phenotype interpretation, we describe stage-specific changes of the palate, heart and gut, and provide examples in which correct diagnosis of malformations relies on correct staging.
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http://dx.doi.org/10.1111/joa.12590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5382591PMC
May 2017

Highly variable penetrance of abnormal phenotypes in embryonic lethal knockout mice.

Wellcome Open Res 2016 27;1. Epub 2017 Feb 27.

Division of Anatomy, Center for Anatomy & Cell Biology, Medical University of Vienna, Wien, Austria.

Identifying genes that are essential for mouse embryonic development and survival through term is a powerful and unbiased way to discover possible genetic determinants of human developmental disorders. Characterising the changes in mouse embryos that result from ablation of lethal genes is a necessary first step towards uncovering their role in normal embryonic development and establishing any correlates amongst human congenital abnormalities. Here we present results gathered to date in the Deciphering the Mechanisms of Developmental Disorders (DMDD) programme, cataloguing the morphological defects identified from comprehensive imaging of 220 homozygous mutant and 114 wild type embryos from 42 lethal and subviable lines, analysed at E14.5. Virtually all mutant embryos show multiple abnormal phenotypes and amongst the 42 lines these affect most organ systems. Within each mutant line, the phenotypes of individual embryos form distinct but overlapping sets. Subcutaneous edema, malformations of the heart or great vessels, abnormalities in forebrain morphology and the musculature of the eyes are all prevalent phenotypes, as is loss or abnormal size of the hypoglossal nerve. Overall, the most striking finding is that no matter how profound the malformation, each phenotype shows highly variable penetrance within a mutant line. These findings have challenging implications for efforts to identify human disease correlates.
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http://dx.doi.org/10.12688/wellcomeopenres.9899.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5159622PMC
February 2017

High-throughput discovery of novel developmental phenotypes.

Nature 2016 09 14;537(7621):508-514. Epub 2016 Sep 14.

Department of Molecular Physiology and Biophysics, Houston, Texas 77030, USA.

Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.
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http://dx.doi.org/10.1038/nature19356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5295821PMC
September 2016

Genome engineering uncovers 54 evolutionarily conserved and testis-enriched genes that are not required for male fertility in mice.

Proc Natl Acad Sci U S A 2016 07 29;113(28):7704-10. Epub 2016 Jun 29.

Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030; Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030; Center for Reproductive Medicine, Baylor College of Medicine, Houston, TX 77030; Center for Drug Discovery, Baylor College of Medicine, Houston, TX 77030; Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030; Department of Pharmacology, Baylor College of Medicine, Houston, TX 77030

Gene-expression analysis studies from Schultz et al. estimate that more than 2,300 genes in the mouse genome are expressed predominantly in the male germ line. As of their 2003 publication [Schultz N, Hamra FK, Garbers DL (2003) Proc Natl Acad Sci USA 100(21):12201-12206], the functions of the majority of these testis-enriched genes during spermatogenesis and fertilization were largely unknown. Since the study by Schultz et al., functional analysis of hundreds of reproductive-tract-enriched genes have been performed, but there remain many testis-enriched genes for which their relevance to reproduction remain unexplored or unreported. Historically, a gene knockout is the "gold standard" to determine whether a gene's function is essential in vivo. Although knockout mice without apparent phenotypes are rarely published, these knockout mouse lines and their phenotypic information need to be shared to prevent redundant experiments. Herein, we used bioinformatic and experimental approaches to uncover mouse testis-enriched genes that are evolutionarily conserved in humans. We then used gene-disruption approaches, including Knockout Mouse Project resources (targeting vectors and mice) and CRISPR/Cas9, to mutate and quickly analyze the fertility of these mutant mice. We discovered that 54 mutant mouse lines were fertile. Thus, despite evolutionary conservation of these genes in vertebrates and in some cases in all eukaryotes, our results indicate that these genes are not individually essential for male mouse fertility. Our phenotypic data are highly relevant in this fiscally tight funding period and postgenomic age when large numbers of genomes are being analyzed for disease association, and will prevent unnecessary expenditures and duplications of effort by others.
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http://dx.doi.org/10.1073/pnas.1608458113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4948324PMC
July 2016

Deficiency of the zinc finger protein ZFP106 causes motor and sensory neurodegeneration.

Hum Mol Genet 2016 Jan 24;25(2):291-307. Epub 2015 Nov 24.

MRC Mammalian Genetics Unit, Harwell, Oxfordshire OX11 0RD, UK,

Zinc finger motifs are distributed amongst many eukaryotic protein families, directing nucleic acid-protein and protein-protein interactions. Zinc finger protein 106 (ZFP106) has previously been associated with roles in immune response, muscle differentiation, testes development and DNA damage, although little is known about its specific function. To further investigate the function of ZFP106, we performed an in-depth characterization of Zfp106 deficient mice (Zfp106(-/-)), and we report a novel role for ZFP106 in motor and sensory neuronal maintenance and survival. Zfp106(-/-) mice develop severe motor abnormalities, major deficits in muscle strength and histopathological changes in muscle. Intriguingly, despite being highly expressed throughout the central nervous system, Zfp106(-/-) mice undergo selective motor and sensory neuronal and axonal degeneration specific to the spinal cord and peripheral nervous system. Neurodegeneration does not occur during development of Zfp106(-/-) mice, suggesting that ZFP106 is likely required for the maintenance of mature peripheral motor and sensory neurons. Analysis of embryonic Zfp106(-/-) motor neurons revealed deficits in mitochondrial function, with an inhibition of Complex I within the mitochondrial electron transport chain. Our results highlight a vital role for ZFP106 in sensory and motor neuron maintenance and reveal a novel player in mitochondrial dysfunction and neurodegeneration.
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http://dx.doi.org/10.1093/hmg/ddv471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4706115PMC
January 2016

A gene expression resource generated by genome-wide lacZ profiling in the mouse.

Dis Model Mech 2015 Nov 20;8(11):1467-78. Epub 2015 Aug 20.

Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK

Knowledge of the expression profile of a gene is a critical piece of information required to build an understanding of the normal and essential functions of that gene and any role it may play in the development or progression of disease. High-throughput, large-scale efforts are on-going internationally to characterise reporter-tagged knockout mouse lines. As part of that effort, we report an open access adult mouse expression resource, in which the expression profile of 424 genes has been assessed in up to 47 different organs, tissues and sub-structures using a lacZ reporter gene. Many specific and informative expression patterns were noted. Expression was most commonly observed in the testis and brain and was most restricted in white adipose tissue and mammary gland. Over half of the assessed genes presented with an absent or localised expression pattern (categorised as 0-10 positive structures). A link between complexity of expression profile and viability of homozygous null animals was observed; inactivation of genes expressed in ≥ 21 structures was more likely to result in reduced viability by postnatal day 14 compared with more restricted expression profiles. For validation purposes, this mouse expression resource was compared with Bgee, a federated composite of RNA-based expression data sets. Strong agreement was observed, indicating a high degree of specificity in our data. Furthermore, there were 1207 observations of expression of a particular gene in an anatomical structure where Bgee had no data, indicating a large amount of novelty in our data set. Examples of expression data corroborating and extending genotype-phenotype associations and supporting disease gene candidacy are presented to demonstrate the potential of this powerful resource.
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http://dx.doi.org/10.1242/dmm.021238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4631787PMC
November 2015

Analysis of mammalian gene function through broad-based phenotypic screens across a consortium of mouse clinics.

Nat Genet 2015 Sep 27;47(9):969-978. Epub 2015 Jul 27.

Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany.

The function of the majority of genes in the mouse and human genomes remains unknown. The mouse embryonic stem cell knockout resource provides a basis for the characterization of relationships between genes and phenotypes. The EUMODIC consortium developed and validated robust methodologies for the broad-based phenotyping of knockouts through a pipeline comprising 20 disease-oriented platforms. We developed new statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no previous functional annotation. We captured data from over 27,000 mice, finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. New phenotypes were uncovered for many genes with previously unknown function, providing a powerful basis for hypothesis generation and further investigation in diverse systems.
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http://dx.doi.org/10.1038/ng.3360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4564951PMC
September 2015

Blastocyst genotyping for quality control of mouse mutant archives: an ethical and economical approach.

Transgenic Res 2015 Oct 16;24(5):921-7. Epub 2015 Jul 16.

Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, CB10 1SA, UK.

With the advent of modern developmental biology and molecular genetics, the scientific community has generated thousands of newly genetically altered strains of laboratory mice with the aim of elucidating gene function. To this end, a large group of Institutions which form the International Mouse Phenotyping Consortium is generating and phenotyping a knockout mouse strain for each of the ~20,000 protein-coding genes using the mutant ES cell resource produced by the International Knockout Mouse Consortium. These strains are made available to the research community via public repositories, mostly as cryopreserved sperm or embryos. To ensure the quality of this frozen resource there is a requirement that for each strain the frozen sperm/embryos are proven able to produce viable mutant progeny, before the live animal resource is removed from cages. Given the current requirement to generate live pups to demonstrate their mutant genotype, this quality control check necessitates the use and generation of many animals and requires considerable time, cage space, technical and economic resources. Here, we describe a simple and efficient method of genotyping pre-implantation stage blastocysts with significant ethical and economic advantages especially beneficial for current and future large-scale mouse mutagenesis projects.
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http://dx.doi.org/10.1007/s11248-015-9897-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4569667PMC
October 2015

Identification of genes important for cutaneous function revealed by a large scale reverse genetic screen in the mouse.

PLoS Genet 2014 Oct 23;10(10):e1004705. Epub 2014 Oct 23.

Department of Biochemistry and Molecular Biology, Monash University, Clayton, Melbourne, Australia; Department of Anatomy and Developmental Biology, Monash University, Clayton, Melbourne, Australia.

The skin is a highly regenerative organ which plays critical roles in protecting the body and sensing its environment. Consequently, morbidity and mortality associated with skin defects represent a significant health issue. To identify genes important in skin development and homeostasis, we have applied a high throughput, multi-parameter phenotype screen to the conditional targeted mutant mice generated by the Wellcome Trust Sanger Institute's Mouse Genetics Project (Sanger-MGP). A total of 562 different mouse lines were subjected to a variety of tests assessing cutaneous expression, macroscopic clinical disease, histological change, hair follicle cycling, and aberrant marker expression. Cutaneous lesions were associated with mutations in 23 different genes. Many of these were not previously associated with skin disease in the organ (Mysm1, Vangl1, Trpc4ap, Nom1, Sparc, Farp2, and Prkab1), while others were ascribed new cutaneous functions on the basis of the screening approach (Krt76, Lrig1, Myo5a, Nsun2, and Nf1). The integration of these skin specific screening protocols into the Sanger-MGP primary phenotyping pipelines marks the largest reported reverse genetic screen undertaken in any organ and defines approaches to maximise the productivity of future projects of this nature, while flagging genes for further characterisation.
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http://dx.doi.org/10.1371/journal.pgen.1004705DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4207618PMC
October 2014

Disruption of the potassium channel regulatory subunit KCNE2 causes iron-deficient anemia.

Exp Hematol 2014 Dec 12;42(12):1053-8.e1. Epub 2014 Aug 12.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, United Kingdom. Electronic address:

Iron homeostasis is a dynamic process that is tightly controlled to balance iron uptake, storage, and export. Reduction of dietary iron from the ferric to the ferrous form is required for uptake by solute carrier family 11 (proton-coupled divalent metal ion transporters), member 2 (Slc11a2) into the enterocytes. Both processes are proton dependent and have led to the suggestion of the importance of acidic gastric pH for the absorption of dietary iron. Potassium voltage-gated channel subfamily E, member 2 (KCNE2), in combination with potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1), form a gastric potassium channel essential for gastric acidification. Deficiency of either Kcne2 or Kcnq1 results in achlorhydia, gastric hyperplasia, and neoplasia, but the impact on iron absorption has not, to our knowledge, been investigated. Here we report that Kcne2-deficient mice, in addition to the previously reported phenotypes, also present with iron-deficient anemia. Interestingly, impaired function of KCNQ1 results in iron-deficient anemia in Jervell and Lange-Nielsen syndrome patients. We speculate that impaired function of KCNE2 could result in the same clinical phenotype.
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http://dx.doi.org/10.1016/j.exphem.2014.07.269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4271779PMC
December 2014

Novel skin phenotypes revealed by a genome-wide mouse reverse genetic screen.

Nat Commun 2014 Apr 11;5:3540. Epub 2014 Apr 11.

Centre for Stem Cells and Regenerative Medicine, King's College London, Guy's Hospital, London SE1 9RT, UK.

Permanent stop-and-shop large-scale mouse mutant resources provide an excellent platform to decipher tissue phenogenomics. Here we analyse skin from 538 knockout mouse mutants generated by the Sanger Institute Mouse Genetics Project. We optimize immunolabelling of tail epidermal wholemounts to allow systematic annotation of hair follicle, sebaceous gland and interfollicular epidermal abnormalities using ontology terms from the Mammalian Phenotype Ontology. Of the 50 mutants with an epidermal phenotype, 9 map to human genetic conditions with skin abnormalities. Some mutant genes are expressed in the skin, whereas others are not, indicating systemic effects. One phenotype is affected by diet and several are incompletely penetrant. In-depth analysis of three mutants, Krt76, Myo5a (a model of human Griscelli syndrome) and Mysm1, provides validation of the screen. Our study is the first large-scale genome-wide tissue phenotype screen from the International Knockout Mouse Consortium and provides an open access resource for the scientific community.
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http://dx.doi.org/10.1038/ncomms4540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3996542PMC
April 2014

Targeting of Slc25a21 is associated with orofacial defects and otitis media due to disrupted expression of a neighbouring gene.

PLoS One 2014 18;9(3):e91807. Epub 2014 Mar 18.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, United Kingdom.

Homozygosity for Slc25a21(tm1a(KOMP)Wtsi) results in mice exhibiting orofacial abnormalities, alterations in carpal and rugae structures, hearing impairment and inflammation in the middle ear. In humans it has been hypothesised that the 2-oxoadipate mitochondrial carrier coded by SLC25A21 may be involved in the disease 2-oxoadipate acidaemia. Unexpectedly, no 2-oxoadipate acidaemia-like symptoms were observed in animals homozygous for Slc25a21(tm1a(KOMP)Wtsi) despite confirmation that this allele reduces Slc25a21 expression by 71.3%. To study the complete knockout, an allelic series was generated using the loxP and FRT sites typical of a Knockout Mouse Project allele. After removal of the critical exon and neomycin selection cassette, Slc25a21 knockout mice homozygous for the Slc25a21(tm1b(KOMP)Wtsi) and Slc25a21(tm1d(KOMP)Wtsi) alleles were phenotypically indistinguishable from wild-type. This led us to explore the genomic environment of Slc25a21 and to discover that expression of Pax9, located 3' of the target gene, was reduced in homozygous Slc25a21(tm1a(KOMP)Wtsi) mice. We hypothesize that the presence of the selection cassette is the cause of the down regulation of Pax9 observed. The phenotypes we observed in homozygous Slc25a21(tm1a(KOMP)Wtsi) mice were broadly consistent with a hypomorphic Pax9 allele with the exception of otitis media and hearing impairment which may be a novel consequence of Pax9 down regulation. We explore the ramifications associated with this particular targeted mutation and emphasise the need to interpret phenotypes taking into consideration all potential underlying genetic mechanisms.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0091807PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3958370PMC
December 2014

Rapid conversion of EUCOMM/KOMP-CSD alleles in mouse embryos using a cell-permeable Cre recombinase.

Transgenic Res 2014 Feb 7;23(1):177-85. Epub 2013 Nov 7.

The Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, CB10 1SA, UK,

We describe here use of a cell-permeable Cre to efficiently convert the EUCOMM/KOMP-CSD tm1a allele to the tm1b form in preimplantation mouse embryos in a high-throughput manner, consistent with the requirements of the International Mouse Phenotyping Consortium-affiliated NIH KOMP2 project. This method results in rapid allele conversion and minimizes the use of experimental animals when compared to conventional Cre transgenic mouse breeding, resulting in a significant reduction in costs and time with increased welfare benefits.
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http://dx.doi.org/10.1007/s11248-013-9764-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3890051PMC
February 2014

Molecular characterization of mutant mouse strains generated from the EUCOMM/KOMP-CSD ES cell resource.

Mamm Genome 2013 Aug 4;24(7-8):286-94. Epub 2013 Aug 4.

The Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, CB10 1SA, UK.

The Sanger Mouse Genetics Project generates knockout mice strains using the EUCOMM/KOMP-CSD embryonic stem (ES) cell collection and characterizes the consequences of the mutations using a high-throughput primary phenotyping screen. Upon achieving germline transmission, new strains are subject to a panel of quality control (QC) PCR- and qPCR-based assays to confirm the correct targeting, cassette structure, and the presence of the 3' LoxP site (required for the potential conditionality of the allele). We report that over 86 % of the 731 strains studied showed the correct targeting and cassette structure, of which 97 % retained the 3' LoxP site. We discuss the characteristics of the lines that failed QC and postulate that the majority of these may be due to mixed ES cell populations which were not detectable with the original screening techniques employed when creating the ES cell resource.
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http://dx.doi.org/10.1007/s00335-013-9467-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3745610PMC
August 2013

A comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N mouse strains.

Genome Biol 2013 Jul 31;14(7):R82. Epub 2013 Jul 31.

Background: The mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as the International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms.

Results: We undertake genome sequence comparisons of C57BL/6J and C57BL/6N to identify SNPs, indels and structural variants, with a focus on identifying all coding variants. We annotate 34 SNPs and 2 indels that distinguish C57BL/6J and C57BL/6N coding sequences, as well as 15 structural variants that overlap a gene. In parallel we assess the comparative phenotypes of the two inbred lines utilizing the EMPReSSslim phenotyping pipeline, a broad based assessment encompassing diverse biological systems. We perform additional secondary phenotyping assessments to explore other phenotype domains and to elaborate phenotype differences identified in the primary assessment. We uncover significant phenotypic differences between the two lines, replicated across multiple centers, in a number of physiological, biochemical and behavioral systems.

Conclusions: Comparison of C57BL/6J and C57BL/6N demonstrates a range of phenotypic differences that have the potential to impact upon penetrance and expressivity of mutational effects in these strains. Moreover, the sequence variants we identify provide a set of candidate genes for the phenotypic differences observed between the two strains.
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http://dx.doi.org/10.1186/gb-2013-14-7-r82DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4053787PMC
July 2013

Genome-wide generation and systematic phenotyping of knockout mice reveals new roles for many genes.

Cell 2013 Jul;154(2):452-64

Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.

Mutations in whole organisms are powerful ways of interrogating gene function in a realistic context. We describe a program, the Sanger Institute Mouse Genetics Project, that provides a step toward the aim of knocking out all genes and screening each line for a broad range of traits. We found that hitherto unpublished genes were as likely to reveal phenotypes as known genes, suggesting that novel genes represent a rich resource for investigating the molecular basis of disease. We found many unexpected phenotypes detected only because we screened for them, emphasizing the value of screening all mutants for a wide range of traits. Haploinsufficiency and pleiotropy were both surprisingly common. Forty-two percent of genes were essential for viability, and these were less likely to have a paralog and more likely to contribute to a protein complex than other genes. Phenotypic data and more than 900 mutants are openly available for further analysis. PAPERCLIP:
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http://dx.doi.org/10.1016/j.cell.2013.06.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3717207PMC
July 2013

High-fat feeding rapidly induces obesity and lipid derangements in C57BL/6N mice.

Mamm Genome 2013 Jun 28;24(5-6):240-51. Epub 2013 May 28.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, UK.

C57BL/6N (B6N) is becoming the standard background for genetic manipulation of the mouse genome. The B6N, whose genome is very closely related to the reference C57BL/6J genome, is versatile in a wide range of phenotyping and experimental settings and large repositories of B6N ES cells have been developed. Here, we present a series of studies showing the baseline characteristics of B6N fed a high-fat diet (HFD) for up to 12 weeks. We show that HFD-fed B6N mice show increased weight gain, fat mass, and hypercholesterolemia compared to control diet-fed mice. In addition, HFD-fed B6N mice display a rapid onset of lipid accumulation in the liver with both macro- and microvacuolation, which became more severe with increasing duration of HFD. Our results suggest that the B6N mouse strain is a versatile background for studying diet-induced metabolic syndrome and may also represent a model for early nonalcoholic fatty liver disease.
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http://dx.doi.org/10.1007/s00335-013-9456-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3685703PMC
June 2013