Publications by authors named "Joanna Bottomley"

11 Publications

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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

An Orphan CpG Island Drives Expression of a miRNA Precursor with an Important Role in Mouse Development.

Epigenomes 2019 Mar 13;3(1). Epub 2019 Mar 13.

Wellcome Centre for Cell Biology, University of Edinburgh, Michael Swann Building, Max Born Crescent, Mayfield Road, Edinburgh EH9 3BF, UK.

Most human genes are associated with promoters embedded in non-methylated, G + C-rich CpG islands (CGIs). Not all CGIs are found at annotated promoters, however, raising the possibility that many serve as promoters for transcripts that do not code for proteins. To test this hypothesis, we searched for novel transcripts in embryonic stem cells (ESCs) that originate within orphan CGIs. Among several candidates, we detected a transcript that included three members of the micro-RNA family: and . Deletion of the CGI prevented expression of the precursor RNA and depleted the included miRNAs. Mice homozygous for this mutation were sub-viable and showed growth and other defects. The results suggest that despite the identity of their seed sequences, members of the miRNA family exert distinct functions that cannot be complemented by other members.
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http://dx.doi.org/10.3390/epigenomes3010007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6660310PMC
March 2019

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

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

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

The mammalian gene function resource: the International Knockout Mouse Consortium.

Mamm Genome 2012 Oct 12;23(9-10):580-6. Epub 2012 Sep 12.

The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1HH, UK.

In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed high-throughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research.
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http://dx.doi.org/10.1007/s00335-012-9422-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3463800PMC
October 2012

Mouse large-scale phenotyping initiatives: overview of the European Mouse Disease Clinic (EUMODIC) and of the Wellcome Trust Sanger Institute Mouse Genetics Project.

Mamm Genome 2012 Oct 9;23(9-10):600-10. Epub 2012 Sep 9.

Institut Clinique de la Souris, PHENOMIN, IGBMC/ICS-MCI, CNRS, INSERM, Université de Strasbourg, UMR7104, UMR964, 1 rue Laurent Fries, 67404 Illkirch, France.

Two large-scale phenotyping efforts, the European Mouse Disease Clinic (EUMODIC) and the Wellcome Trust Sanger Institute Mouse Genetics Project (SANGER-MGP), started during the late 2000s with the aim to deliver a comprehensive assessment of phenotypes or to screen for robust indicators of diseases in mouse mutants. They both took advantage of available mouse mutant lines but predominantly of the embryonic stem (ES) cells resources derived from the European Conditional Mouse Mutagenesis programme (EUCOMM) and the Knockout Mouse Project (KOMP) to produce and study 799 mouse models that were systematically analysed with a comprehensive set of physiological and behavioural paradigms. They captured more than 400 variables and an additional panel of metadata describing the conditions of the tests. All the data are now available through EuroPhenome database (www.europhenome.org) and the WTSI mouse portal (http://www.sanger.ac.uk/mouseportal/), and the corresponding mouse lines are available through the European Mouse Mutant Archive (EMMA), the International Knockout Mouse Consortium (IKMC), or the Knockout Mouse Project (KOMP) Repository. Overall conclusions from both studies converged, with at least one phenotype scored in at least 80% of the mutant lines. In addition, 57% of the lines were viable, 13% subviable, 30% embryonic lethal, and 7% displayed fertility impairments. These efforts provide an important underpinning for a future global programme that will undertake the complete functional annotation of the mammalian genome in the mouse model.
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http://dx.doi.org/10.1007/s00335-012-9418-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3463797PMC
October 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

A series of 2(Z)-2-benzylidene-6,7-dihydroxybenzofuran-3[2H]-ones as inhibitors of chorismate synthase.

Bioorg Med Chem Lett 2003 Feb;13(3):423-6

PanTherix Ltd., Todd Campus, West of Scotland Science Park, Glasgow G20 0XA, Scotland, UK.

A series of 2(Z)-2-benzylidene-6,7-dihydroxybenzofuran-3[2H]-ones was identified as potent inhibitors of bacterial chorismate synthase. The 2'-hydroxy-4'-pentoxy analogue 33 is a potent inhibitor of Streptococcus pneumoniae chorismate synthase.
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http://dx.doi.org/10.1016/s0960-894x(02)00957-5DOI Listing
February 2003