Publications by authors named "Edward Ryder"

33 Publications

High-throughput genotyping of high-homology mutant mouse strains by next-generation sequencing.

Methods 2021 07 20;191:78-86. Epub 2020 Oct 20.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SA, UK. Electronic address:

Genotyping of knockout alleles in mice is commonly performed by end-point PCR or gene-specific/universal cassette qPCR. Both have advantages and limitations in terms of assay design and interpretation of results. As an alternative method for high-throughput genotyping, we investigated next generation sequencing (NGS) of PCR amplicons, with a focus on CRISPR-mediated exon deletions where antibiotic selection markers are not present. By multiplexing the wild type and mutant-specific PCR reactions, the genotype can be called by the relative sequence counts of each product. The system is highly scalable and can be applied to a variety of different allele types, including those produced by the International Mouse Phenotyping Consortium and associated projects. One potential challenge with any assay design is locating unique areas of the genome, especially when working with gene families or regions of high homology. These can result in misleading or ambiguous genotypes for either qPCR or end-point assays. Here, we show that genotyping by NGS can negate these issues by simple, automated filtering of undesired sequences. Analysis and genotype calls can also be fully automated, using FASTQ or FASTA input files and an in-house Perl script and SQL database.
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http://dx.doi.org/10.1016/j.ymeth.2020.10.011DOI Listing
July 2021

Tumors induce de novo steroid biosynthesis in T cells to evade immunity.

Nat Commun 2020 07 17;11(1):3588. Epub 2020 Jul 17.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, CB10 1SA, UK.

Tumors subvert immune cell function to evade immune responses, yet the complex mechanisms driving immune evasion remain poorly understood. Here we show that tumors induce de novo steroidogenesis in T lymphocytes to evade anti-tumor immunity. Using a transgenic steroidogenesis-reporter mouse line we identify and characterize de novo steroidogenic immune cells, defining the global gene expression identity of these steroid-producing immune cells and gene regulatory networks by using single-cell transcriptomics. Genetic ablation of T cell steroidogenesis restricts primary tumor growth and metastatic dissemination in mouse models. Steroidogenic T cells dysregulate anti-tumor immunity, and inhibition of the steroidogenesis pathway is sufficient to restore anti-tumor immunity. This study demonstrates T cell de novo steroidogenesis as a mechanism of anti-tumor immunosuppression and a potential druggable target.
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http://dx.doi.org/10.1038/s41467-020-17339-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7368057PMC
July 2020

Common and distinct transcriptional signatures of mammalian embryonic lethality.

Nat Commun 2019 06 26;10(1):2792. Epub 2019 Jun 26.

Wellcome Sanger Institute, Wellcome Genome Campus, Cambridge, CB10 1SA, UK.

The Deciphering the Mechanisms of Developmental Disorders programme has analysed the morphological and molecular phenotypes of embryonic and perinatal lethal mouse mutant lines in order to investigate the causes of embryonic lethality. Here we show that individual whole-embryo RNA-seq of 73 mouse mutant lines (>1000 transcriptomes) identifies transcriptional events underlying embryonic lethality and associates previously uncharacterised genes with specific pathways and tissues. For example, our data suggest that Hmgxb3 is involved in DNA-damage repair and cell-cycle regulation. Further, we separate embryonic delay signatures from mutant line-specific transcriptional changes by developing a baseline mRNA expression catalogue of wild-type mice during early embryogenesis (4-36 somites). Analysis of transcription outside coding sequence identifies deregulation of repetitive elements in Morc2a mutants and a gene involved in gene-specific splicing. Collectively, this work provides a large scale resource to further our understanding of early embryonic developmental disorders.
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http://dx.doi.org/10.1038/s41467-019-10642-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594971PMC
June 2019

FBXO7 sensitivity of phenotypic traits elucidated by a hypomorphic allele.

PLoS One 2019 6;14(3):e0212481. Epub 2019 Mar 6.

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

FBXO7 encodes an F box containing protein that interacts with multiple partners to facilitate numerous cellular processes and has a canonical role as part of an SCF E3 ubiquitin ligase complex. Mutation of FBXO7 is responsible for an early onset Parkinsonian pyramidal syndrome and genome-wide association studies have linked variants in FBXO7 to erythroid traits. A putative orthologue in Drosophila, nutcracker, has been shown to regulate the proteasome, and deficiency of nutcracker results in male infertility. Therefore, we reasoned that modulating Fbxo7 levels in a murine model could provide insights into the role of this protein in mammals. We used a targeted gene trap model which retained 4-16% residual gene expression and assessed the sensitivity of phenotypic traits to gene dosage. Fbxo7 hypomorphs showed regenerative anaemia associated with a shorter erythrocyte half-life, and male mice were infertile. Alterations to T cell phenotypes were also observed, which intriguingly were both T cell intrinsic and extrinsic. Hypomorphic mice were also sensitive to infection with Salmonella, succumbing to a normally sublethal challenge. Despite these phenotypes, Fbxo7 hypomorphs were produced at a normal Mendelian ratio with a normal lifespan and no evidence of neurological symptoms. These data suggest that erythrocyte survival, T cell development and spermatogenesis are particularly sensitive to Fbxo7 gene dosage.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0212481PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6402633PMC
November 2019

Mammalian Hbs1L deficiency causes congenital anomalies and developmental delay associated with Pelota depletion and 80S monosome accumulation.

PLoS Genet 2019 02 1;15(2):e1007917. Epub 2019 Feb 1.

Division of Newborn Medicine, Boston Children's Hospital, Boston, Massachusetts, United States of America.

Hbs1 has been established as a central component of the cell's translational quality control pathways in both yeast and prokaryotic models; however, the functional characteristics of its human ortholog (Hbs1L) have not been well-defined. We recently reported a novel human phenotype resulting from a mutation in the critical coding region of the HBS1L gene characterized by facial dysmorphism, severe growth restriction, axial hypotonia, global developmental delay and retinal pigmentary deposits. Here we further characterize downstream effects of the human HBS1L mutation. HBS1L has three transcripts in humans, and RT-PCR demonstrated reduced mRNA levels corresponding with transcripts V1 and V2 whereas V3 expression was unchanged. Western blot analyses revealed Hbs1L protein was absent in the patient cells. Additionally, polysome profiling revealed an abnormal aggregation of 80S monosomes in patient cells under baseline conditions. RNA and ribosomal sequencing demonstrated an increased translation efficiency of ribosomal RNA in Hbs1L-deficient fibroblasts, suggesting that there may be a compensatory increase in ribosome translation to accommodate the increased 80S monosome levels. This enhanced translation was accompanied by upregulation of mTOR and 4-EBP protein expression, suggesting an mTOR-dependent phenomenon. Furthermore, lack of Hbs1L caused depletion of Pelota protein in both patient cells and mouse tissues, while PELO mRNA levels were unaffected. Inhibition of proteasomal function partially restored Pelota expression in human Hbs1L-deficient cells. We also describe a mouse model harboring a knockdown mutation in the murine Hbs1l gene that shared several of the phenotypic elements observed in the Hbs1L-deficient human including facial dysmorphism, growth restriction and retinal deposits. The Hbs1lKO mice similarly demonstrate diminished Pelota levels that were rescued by proteasome inhibition.
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http://dx.doi.org/10.1371/journal.pgen.1007917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6373978PMC
February 2019

No unexpected CRISPR-Cas9 off-target activity revealed by trio sequencing of gene-edited mice.

PLoS Genet 2018 07 9;14(7):e1007503. Epub 2018 Jul 9.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, United Kingdom.

CRISPR-Cas9 technologies have transformed genome-editing of experimental organisms and have immense therapeutic potential. Despite significant advances in our understanding of the CRISPR-Cas9 system, concerns remain over the potential for off-target effects. Recent studies have addressed these concerns using whole-genome sequencing (WGS) of gene-edited embryos or animals to search for de novo mutations (DNMs), which may represent candidate changes introduced by poor editing fidelity. Critically, these studies used strain-matched, but not pedigree-matched controls and thus were unable to reliably distinguish generational or colony-related differences from true DNMs. Here we used a trio design and whole genome sequenced 8 parents and 19 embryos, where 10 of the embryos were mutagenised with well-characterised gRNAs targeting the coat colour Tyrosinase (Tyr) locus. Detailed analyses of these whole genome data allowed us to conclude that if CRISPR mutagenesis were causing SNV or indel off-target mutations in treated embryos, then the number of these mutations is not statistically distinguishable from the background rate of DNMs occurring due to other processes.
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http://dx.doi.org/10.1371/journal.pgen.1007503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6057650PMC
July 2018

Placentation defects are highly prevalent in embryonic lethal mouse mutants.

Nature 2018 03 14;555(7697):463-468. Epub 2018 Mar 14.

The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK.

Large-scale phenotyping efforts have demonstrated that approximately 25-30% of mouse gene knockouts cause intrauterine lethality. Analysis of these mutants has largely focused on the embryo and not the placenta, despite the crucial role of this extraembryonic organ for developmental progression. Here we screened 103 embryonic lethal and sub-viable mouse knockout lines from the Deciphering the Mechanisms of Developmental Disorders program for placental phenotypes. We found that 68% of knockout lines that are lethal at or after mid-gestation exhibited placental dysmorphologies. Early lethality (embryonic days 9.5-14.5) is almost always associated with severe placental malformations. Placental defects correlate strongly with abnormal brain, heart and vascular development. Analysis of mutant trophoblast stem cells and conditional knockouts suggests that a considerable number of factors that cause embryonic lethality when ablated have primary gene function in trophoblast cells. Our data highlight the hugely under-appreciated importance of placental defects in contributing to abnormal embryo development and suggest key molecular nodes that govern placenta formation.
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http://dx.doi.org/10.1038/nature26002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866719PMC
March 2018

Genome-wide in vivo screen identifies novel host regulators of metastatic colonization.

Nature 2017 01 4;541(7636):233-236. Epub 2017 Jan 4.

Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge CB10 1SA, UK.

Metastasis is the leading cause of death for cancer patients. This multi-stage process requires tumour cells to survive in the circulation, extravasate at distant sites, then proliferate; it involves contributions from both the tumour cell and tumour microenvironment ('host', which includes stromal cells and the immune system). Studies suggest the early steps of the metastatic process are relatively efficient, with the post-extravasation regulation of tumour growth ('colonization') being critical in determining metastatic outcome. Here we show the results of screening 810 mutant mouse lines using an in vivo assay to identify microenvironmental regulators of metastatic colonization. We identify 23 genes that, when disrupted in mouse, modify the ability of tumour cells to establish metastatic foci, with 19 of these genes not previously demonstrated to play a role in host control of metastasis. The largest reduction in pulmonary metastasis was observed in sphingosine-1-phosphate (S1P) transporter spinster homologue 2 (Spns2)-deficient mice. We demonstrate a novel outcome of S1P-mediated regulation of lymphocyte trafficking, whereby deletion of Spns2, either globally or in a lymphatic endothelial-specific manner, creates a circulating lymphopenia and a higher percentage of effector T cells and natural killer (NK) cells present in the lung. This allows for potent tumour cell killing, and an overall decreased metastatic burden.
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http://dx.doi.org/10.1038/nature20792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5603286PMC
January 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

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

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

Deubiquitinase MYSM1 Is Essential for Normal Fetal Liver Hematopoiesis and for the Maintenance of Hematopoietic Stem Cells in Adult Bone Marrow.

Stem Cells Dev 2015 Aug 30;24(16):1865-77. Epub 2015 Jun 30.

1 Department of Physiology, McGill University , Montreal, Quebec, Canada .

MYSM1 is a chromatin-interacting deubiquitinase recently shown to be essential for hematopoietic stem cell (HSC) function and normal progression of hematopoiesis in both mice and humans. However, it remains unknown whether the loss of function in Mysm1-deficient HSCs is due to the essential role of MYSM1 in establishing the HSC pool during development or due to a continuous requirement for MYSM1 in adult HSCs. In this study we, for the first time, address these questions first, by performing a detailed analysis of hematopoiesis in the fetal livers of Mysm1-knockout mice, and second, by assessing the effects of an inducible Mysm1 ablation on adult HSC functions. Our data indicate that MYSM1 is essential for normal HSC function and progression of hematopoiesis in the fetal liver. Furthermore, the inducible knockout model demonstrates a continuous requirement for MYSM1 to maintain HSC functions and antagonize p53 activation in adult bone marrow. These studies advance our understanding of the role of MYSM1 in HSC biology, and provide new insights into the human hematopoietic failure syndrome resulting from MYSM1 deficiency.
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http://dx.doi.org/10.1089/scd.2015.0058DOI Listing
August 2015

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

Genomic analysis of a novel spontaneous albino C57BL/6N mouse strain.

Genesis 2013 Jul 25;51(7):523-8. Epub 2013 May 25.

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

We report an albino C57BL/6N mouse strain carrying a spontaneous mutation in the tyrosinase gene (C57BL/6N-Tyr(cWTSI)). Deep whole genome sequencing of founder mice revealed very little divergence from C57BL/6NJ and C57BL/6N (Taconic). This coisogenic strain will be of great utility for the International Mouse Phenotyping Consortium (IMPC), which uses the EUCOMM/KOMP targeted C57BL/6N ES cell resource, and other investigators wishing to work on a defined C57BL/6N background.
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http://dx.doi.org/10.1002/dvg.22398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3799019PMC
July 2013

Deficiency for the ubiquitin ligase UBE3B in a blepharophimosis-ptosis-intellectual-disability syndrome.

Am J Hum Genet 2012 Dec 29;91(6):998-1010. Epub 2012 Nov 29.

Raphael Recanati Genetics Institute, Rabin Medical Center, Beilinson Campus, Petah Tikva 49100, Israel.

Ubiquitination plays a crucial role in neurodevelopment as exemplified by Angelman syndrome, which is caused by genetic alterations of the ubiquitin ligase-encoding UBE3A gene. Although the function of UBE3A has been widely studied, little is known about its paralog UBE3B. By using exome and capillary sequencing, we here identify biallelic UBE3B mutations in four patients from three unrelated families presenting an autosomal-recessive blepharophimosis-ptosis-intellectual-disability syndrome characterized by developmental delay, growth retardation with a small head circumference, facial dysmorphisms, and low cholesterol levels. UBE3B encodes an uncharacterized E3 ubiquitin ligase. The identified UBE3B variants include one frameshift and two splice-site mutations as well as a missense substitution affecting the highly conserved HECT domain. Disruption of mouse Ube3b leads to reduced viability and recapitulates key aspects of the human disorder, such as reduced weight and brain size and a downregulation of cholesterol synthesis. We establish that the probable Caenorhabditis elegans ortholog of UBE3B, oxi-1, functions in the ubiquitin/proteasome system in vivo and is especially required under oxidative stress conditions. Our data reveal the pleiotropic effects of UBE3B deficiency and reinforce the physiological importance of ubiquitination in neuronal development and function in mammals.
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http://dx.doi.org/10.1016/j.ajhg.2012.10.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3516591PMC
December 2012

Large-scale mouse knockouts and phenotypes.

Wiley Interdiscip Rev Syst Biol Med 2012 Nov-Dec;4(6):547-63. Epub 2012 Aug 15.

Mouse Pipelines, Wellcome Trust Sanger Institute, Hinxton, UK.

Standardized phenotypic analysis of mutant forms of every gene in the mouse genome will provide fundamental insights into mammalian gene function and advance human and animal health. The availability of the human and mouse genome sequences, the development of embryonic stem cell mutagenesis technology, the standardization of phenotypic analysis pipelines, and the paradigm-shifting industrialization of these processes have made this a realistic and achievable goal. The size of this enterprise will require global coordination to ensure economies of scale in both the generation and primary phenotypic analysis of the mutant strains, and to minimize unnecessary duplication of effort. To provide more depth to the functional annotation of the genome, effective mechanisms will also need to be developed to disseminate the information and resources produced to the wider community. Better models of disease, potential new drug targets with novel mechanisms of action, and completely unsuspected genotype-phenotype relationships covering broad aspects of biology will become apparent. To reach these goals, solutions to challenges in mouse production and distribution, as well as development of novel, ever more powerful phenotypic analysis modalities will be necessary. It is a challenging and exciting time to work in mouse genetics.
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http://dx.doi.org/10.1002/wsbm.1183DOI Listing
February 2013

Rapid-throughput skeletal phenotyping of 100 knockout mice identifies 9 new genes that determine bone strength.

PLoS Genet 2012 2;8(8):e1002858. Epub 2012 Aug 2.

Molecular Endocrinology Group, Department of Medicine, Imperial College London, London, United Kingdom.

Osteoporosis is a common polygenic disease and global healthcare priority but its genetic basis remains largely unknown. We report a high-throughput multi-parameter phenotype screen to identify functionally significant skeletal phenotypes in mice generated by the Wellcome Trust Sanger Institute Mouse Genetics Project and discover novel genes that may be involved in the pathogenesis of osteoporosis. The integrated use of primary phenotype data with quantitative x-ray microradiography, micro-computed tomography, statistical approaches and biomechanical testing in 100 unselected knockout mouse strains identified nine new genetic determinants of bone mass and strength. These nine new genes include five whose deletion results in low bone mass and four whose deletion results in high bone mass. None of the nine genes have been implicated previously in skeletal disorders and detailed analysis of the biomechanical consequences of their deletion revealed a novel functional classification of bone structure and strength. The organ-specific and disease-focused strategy described in this study can be applied to any biological system or tractable polygenic disease, thus providing a general basis to define gene function in a system-specific manner. Application of the approach to diseases affecting other physiological systems will help to realize the full potential of the International Mouse Phenotyping Consortium.
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http://dx.doi.org/10.1371/journal.pgen.1002858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3410859PMC
December 2012

The role of sphingosine-1-phosphate transporter Spns2 in immune system function.

J Immunol 2012 Jul 4;189(1):102-11. Epub 2012 Jun 4.

Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, United Kingdom.

Sphingosine-1-phosphate (S1P) is lipid messenger involved in the regulation of embryonic development, immune system functions, and many other physiological processes. However, the mechanisms of S1P transport across cellular membranes remain poorly understood, with several ATP-binding cassette family members and the spinster 2 (Spns2) member of the major facilitator superfamily known to mediate S1P transport in cell culture. Spns2 was also shown to control S1P activities in zebrafish in vivo and to play a critical role in zebrafish cardiovascular development. However, the in vivo roles of Spns2 in mammals and its involvement in the different S1P-dependent physiological processes have not been investigated. In this study, we characterized Spns2-null mouse line carrying the Spns2(tm1a(KOMP)Wtsi) allele (Spns2(tm1a)). The Spns2(tm1a/tm1a) animals were viable, indicating a divergence in Spns2 function from its zebrafish ortholog. However, the immunological phenotype of the Spns2(tm1a/tm1a) mice closely mimicked the phenotypes of partial S1P deficiency and impaired S1P-dependent lymphocyte trafficking, with a depletion of lymphocytes in circulation, an increase in mature single-positive T cells in the thymus, and a selective reduction in mature B cells in the spleen and bone marrow. Spns2 activity in the nonhematopoietic cells was critical for normal lymphocyte development and localization. Overall, Spns2(tm1a/tm1a) resulted in impaired humoral immune responses to immunization. This study thus demonstrated a physiological role for Spns2 in mammalian immune system functions but not in cardiovascular development. Other components of the S1P signaling network are investigated as drug targets for immunosuppressive therapy, but the selective action of Spns2 may present an advantage in this regard.
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http://dx.doi.org/10.4049/jimmunol.1200282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3381845PMC
July 2012

In vivo analysis of proteomes and interactomes using Parallel Affinity Capture (iPAC) coupled to mass spectrometry.

Mol Cell Proteomics 2011 Jun 29;10(6):M110.002386. Epub 2011 Mar 29.

Cambridge Centre for Proteomics, University of Cambridge, Cambridge, UK.

Affinity purification coupled to mass spectrometry provides a reliable method for identifying proteins and their binding partners. In this study we have used Drosophila melanogaster proteins triple tagged with Flag, Strep II, and Yellow fluorescent protein in vivo within affinity pull-down experiments and isolated these proteins in their native complexes from embryos. We describe a pipeline for determining interactomes by Parallel Affinity Capture (iPAC) and show its use by identifying partners of several protein baits with a range of sizes and subcellular locations. This purification protocol employs the different tags in parallel and involves detailed comparison of resulting mass spectrometry data sets, ensuring the interaction lists achieved are of high confidence. We show that this approach identifies known interactors of bait proteins as well as novel interaction partners by comparing data achieved with published interaction data sets. The high confidence in vivo protein data sets presented here add new data to the currently incomplete D. melanogaster interactome. Additionally we report contaminant proteins that are persistent with affinity purifications irrespective of the tagged bait.
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http://dx.doi.org/10.1074/mcp.M110.002386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3108830PMC
June 2011

Impaired tissue growth is mediated by checkpoint kinase 1 (CHK1) in the integrated stress response.

J Cell Sci 2010 Sep 3;123(Pt 17):2892-900. Epub 2010 Aug 3.

Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research (CIMR), Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY, UK.

The integrated stress response (ISR) protects cells from numerous forms of stress and is involved in the growth of solid tumours; however, it is unclear how the ISR acts on cellular proliferation. We have developed a model of ISR signalling with which to study its effects on tissue growth. Overexpression of the ISR kinase PERK resulted in a striking atrophic eye phenotype in Drosophila melanogaster that could be rescued by co-expressing the eIF2alpha phosphatase GADD34. A genetic screen of 3000 transposon insertions identified grapes, the gene that encodes the Drosophila orthologue of checkpoint kinase 1 (CHK1). Knockdown of grapes by RNAi rescued eye development despite ongoing PERK activation. In mammalian cells, CHK1 was activated by agents that induce ER stress, which resulted in a G2 cell cycle delay. PERK was both necessary and sufficient for CHK1 activation. These findings indicate that non-genotoxic misfolded protein stress accesses DNA-damage-induced cell cycle checkpoints to couple the ISR to cell cycle arrest.
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http://dx.doi.org/10.1242/jcs.070078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2923568PMC
September 2010

Fenton chemistry and oxidative stress mediate the toxicity of the beta-amyloid peptide in a Drosophila model of Alzheimer's disease.

Eur J Neurosci 2009 Apr 23;29(7):1335-47. Epub 2009 Mar 23.

Department of Medicine, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Cambridge, UK.

The mechanism by which aggregates of the beta-amyloid peptide (Abeta) mediate their toxicity is uncertain. We show here that the expression of the 42-amino-acid isoform of Abeta (Abeta(1-42)) changes the expression of genes involved in oxidative stress in a Drosophila model of Alzheimer's disease. A subsequent genetic screen confirmed the importance of oxidative stress and a molecular dissection of the steps in the cellular metabolism of reactive oxygen species revealed that the iron-binding protein ferritin and the H(2)O(2) scavenger catalase are the most potent suppressors of the toxicity of wild-type and Arctic (E22G) Abeta(1-42). Likewise, treatment with the iron-binding compound clioquinol increased the lifespan of flies expressing Arctic Abeta(1-42). The effect of iron appears to be mediated by oxidative stress as ferritin heavy chain co-expression reduced carbonyl levels in Abeta(1-42) flies by 65% and restored the survival and locomotion function to normal. This was achieved despite the presence of elevated levels of the Abeta(1-42). Taken together, our data show that oxidative stress, probably mediated by the hydroxyl radical and generated by the Fenton reaction, is essential for Abeta(1-42) toxicity in vivo and provide strong support for Alzheimer's disease therapies based on metal chelation.
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http://dx.doi.org/10.1111/j.1460-9568.2009.06701.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2777252PMC
April 2009

The evolution of the DLK1-DIO3 imprinted domain in mammals.

PLoS Biol 2008 Jun;6(6):e135

Department of Physiology, Development, and Neuroscience, University of Cambridge, Cambridge, United Kingdom.

A comprehensive, domain-wide comparative analysis of genomic imprinting between mammals that imprint and those that do not can provide valuable information about how and why imprinting evolved. The imprinting status, DNA methylation, and genomic landscape of the Dlk1-Dio3 cluster were determined in eutherian, metatherian, and prototherian mammals including tammar wallaby and platypus. Imprinting across the whole domain evolved after the divergence of eutherian from marsupial mammals and in eutherians is under strong purifying selection. The marsupial locus at 1.6 megabases, is double that of eutherians due to the accumulation of LINE repeats. Comparative sequence analysis of the domain in seven vertebrates determined evolutionary conserved regions common to particular sub-groups and to all vertebrates. The emergence of Dlk1-Dio3 imprinting in eutherians has occurred on the maternally inherited chromosome and is associated with region-specific resistance to expansion by repetitive elements and the local introduction of noncoding transcripts including microRNAs and C/D small nucleolar RNAs. A recent mammal-specific retrotransposition event led to the formation of a completely new gene only in the eutherian domain, which may have driven imprinting at the cluster.
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http://dx.doi.org/10.1371/journal.pbio.0060135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2408620PMC
June 2008

The DrosDel deletion collection: a Drosophila genomewide chromosomal deficiency resource.

Genetics 2007 Sep 24;177(1):615-29. Epub 2007 Aug 24.

Department of Genetics, University of Cambridge, Cambridge CB2 3EH, United Kingdom.

We describe a second-generation deficiency kit for Drosophila melanogaster composed of molecularly mapped deletions on an isogenic background, covering approximately 77% of the Release 5.1 genome. Using a previously reported collection of FRT-bearing P-element insertions, we have generated 655 new deletions and verified a set of 209 deletion-bearing fly stocks. In addition to deletions, we demonstrate how the P elements may also be used to generate a set of custom inversions and duplications, particularly useful for balancing difficult regions of the genome carrying haplo-insufficient loci. We describe a simple computational resource that facilitates selection of appropriate elements for generating custom deletions. Finally, we provide a computational resource that facilitates selection of other mapped FRT-bearing elements that, when combined with the DrosDel collection, can theoretically generate over half a million precisely mapped deletions.
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http://dx.doi.org/10.1534/genetics.107.076216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2013729PMC
September 2007

Host Resistance to Mirafiori lettuce big-vein virus and Lettuce big-vein associated virus and Virus Sequence Diversity and Frequency in California.

Plant Dis 2006 Feb;90(2):233-239

United States Department of Agriculture, Agricultural Research Service, Crop Improvement and Protection Unit, 1636 E. Alisal St., Salinas, CA 93905.

Big vein is an economically damaging disease of lettuce (Lactuca sativa) caused by the Olpidium brassicae-vectored Mirafiori lettuce big-vein virus (MLBVV). Lettuce big-vein associated virus (LBVaV) is also frequently identified in symptomatic plants, but no causal relationship has been demonstrated. Although big vein is a perennial problem in the United States, the extent of MLBVV and LBVaV infection and diversity is unknown. Lettuce cultivars partially resistant to big vein reduce losses, but do not eliminate disease. While Lactuca virosa does not develop big vein symptoms, it has not been tested for infection with MLBVV or LBVaV. Lettuce cultivars Great Lakes 65, Pavane, Margarita, and L. virosa accession IVT280 were evaluated for big vein incidence and virus infection in inoculated greenhouse trials. Additional lettuce samples were collected from field sites in California, classified for symptom severity, and evaluated for virus infection. Reverse transcription-polymerase chain reaction and nucleotide sequencing were used to determine infection with MLBVV and LBVaV, and sequence diversity among viral isolates, respectively. Infections with MLBVV and MLBVV/LBVaV were dependent on big vein symptom expression in California production areas, and isolates were closely related to those found in Europe and Japan. Partial big vein resistance was identified in Margarita and Pavane; however, MLBVV infection was found in asymptomatic plants. L. virosa IVT280 remained symptomless and virus free, suggesting that it is immune to MLBVV and LBVaV.
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http://dx.doi.org/10.1094/PD-90-0233DOI Listing
February 2006

Weedborne Reservoirs and Seed Transmission of Verticillium dahliae in Lettuce.

Plant Dis 2005 Mar;89(3):317-324

Department of Plant Pathology, University of California, Davis, c/o USARS, Salinas.

The seed transmission of Verticillium dahliae was evaluated in lettuce (Lactuca sativa). Seed collected from lettuce plants infected with V. dahliae were plated with or without surface sterilization on Sorenson's modified NP10 medium. Of the seed plated with or without surface sterilization, 90 and 66%, respectively, yielded colonies of V. dahliae. The incidence of Verticillium wilt ranged from 55 to 80% among lettuce plants grown from seed harvested from infected plants. All evaluated isolates of V. dahliae were capable of seed transmission in lettuce. A V. tricorpus isolate failed to cause significant disease in lettuce or to become seedborne. Storage of contaminated seed at seven temperatures ranging from -20 to 15°C for up to 72 weeks did not reduce the incidence of V. dahliae in seed, whereas storage at room temperature (23 ± 2°C) for 20 to 52 weeks reduced the incidence of V. dahliae without affecting seed viability. Of the 11 weed species collected from fields with a known history of Verticillium wilt of lettuce, four yielded V. dahliae. Pathogenicity tests demonstrated that isolates of V. dahliae from Sonchus oleraceus, Capsella bursa-pastoris, and Solanum sarrachoides were as virulent as or more virulent than an isolate of V. dahliae from lettuce. These results demonstrate the potential of seedborne and weedborne inoculum to disseminate V. dahliae.
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http://dx.doi.org/10.1094/PD-89-0317DOI Listing
March 2005

Genetic analysis and mapping of resistance to lettuce dieback: a soilborne disease caused by tombusviruses.

Theor Appl Genet 2005 Jan 11;110(2):259-68. Epub 2004 Dec 11.

US Department of Agriculture, Agricultural Research Service, 1636 E. Alisal St., Salinas, CA, 93905, USA.

A diverse collection of modern, heirloom and specialty cultivars, plant introduction (PI) accessions, and breeding lines of lettuce were screened for susceptibility to lettuce dieback, which is a disease caused by soilborne viruses of the family Tombusviridae. Susceptibility was evaluated by visual symptom assessment in fields that had been previously shown to be infested with Lettuce necrotic stunt virus. Of the 241 genotypes tested in multiple field experiments, 76 remained symptom-free in infested fields and were therefore classified as resistant to dieback. Overall, resistant genotypes were as prevalent among modern cultivars as in heirloom cultivars or primitive germplasm. Within modern germplasm, however, all crisphead (iceberg) cultivars were resistant, while all romaine cultivars were susceptible. Using enzyme-linked immunosorbent assay, tombusviruses were detected in leaves of some plants of resistant genotypes that were grown in infested fields, suggesting that symptom-free plants are not immune to viral infection. The inheritance of resistance was studied for 'Salinas', a modern iceberg cultivar, and PI 491224, the progenitor of recently released romaine germplasm with resistance to lettuce dieback. Resistance was conferred by a dominant allele at a single locus in both genotypes. The tombusvirus resistance locus from 'Salinas', Tvr1, was mapped in an intraspecific Lactuca sativa population to a location that corresponds to linkage group 2 on the consensus map of Lactuca. The largest cluster of resistance genes in lettuce, the Dm1/Dm3 cluster, is found on this linkage group; however, the precise position of Tvr1 relative to this cluster has not yet been determined. To our knowledge, Tvr1 is the first tombusvirus resistance gene identified for any plant host.
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http://dx.doi.org/10.1007/s00122-004-1825-3DOI Listing
January 2005