Standardized Welfare Terms for the Zebrafish Community.

This is the final version of the article. It first appeared from Mary Ann Liebert via https://doi.org/10.1089/zeb.2016.1248Managing the welfare of laboratory animals is critical to animal health, vital in the understanding of phenotypes created by treatment or genetic alteration and ensures compliance of regulations. Part of an animal welfare assessment is the requirement to record observations, ensuring all those responsible for the animals are aware of their health status and can act accordingly. Although the use of zebrafish in research continues to increase, guidelines for conducting welfare assessments and the reporting of observations are considered unclear compared to mammalian species. To support the movement of zebrafish between facilities, significant improvement would be achieved through the use of standardized terms to ensure clarity and consistency between facilities. Improving the clarity of terminology around welfare not only addresses our ethical obligation but also supports the research goals and provides a searchable description of the phenotypes. A Collaboration between the Wellcome Trust Sanger Institute and Cambridge University (Department of Medicine-Laboratory of Molecular Biology) has led to the creation of the zebrafish welfare terms from which standardization of terminology can be achieved.This work was supported by the Wellcome Trust (grant no. 098051), the National Institutes of Health Office of Research Infrastructure Programs award (P40OD011021), the National Institutes of Health (grant no. A154503), National Institutes for Health Research to the Biomedical Research Centre (grant no. RG64229), and a Wellcome Trust Personal Research Fellowship (103950/Z/14/Z)

Mouse models of rhinovirus-induced disease and exacerbation of allergic airway inflammation

Rhinoviruses cause serious morbidity and mortality as the major etiological agents of asthma exacerbations and the common cold. A major obstacle to understanding disease pathogenesis and to the development of effective therapies has been the lack of a small-animal model for rhinovirus infection. Of the 100 known rhinovirus serotypes, 90% (the major group) use human intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor and do not bind mouse ICAM-1; the remaining 10% (the minor group) use a member of the low-density lipoprotein receptor family and can bind the mouse counterpart. Here we describe three novel mouse models of rhinovirus infection: minor-group rhinovirus infection of BALB/c mice, major-group rhinovirus infection of transgenic BALB/c mice expressing a mouse-human ICAM-1 chimera and rhinovirus-induced exacerbation of allergic airway inflammation. These models have features similar to those observed in rhinovirus infection in humans, including augmentation of allergic airway inflammation, and will be useful in the development of future therapies for colds and asthma exacerbations

Diversity Arrays Technology (DArT) for Pan-Genomic Evolutionary Studies of Non-Model Organisms

Background: High-throughput tools for pan-genomic study, especially the DNA microarray platform, have sparked a remarkable increase in data production and enabled a shift in the scale at which biological investigation is possible. The use of microarrays to examine evolutionary relationships and processes, however, is predominantly restricted to model or near-model organisms. Methodology/Principal Findings: This study explores the utility of Diversity Arrays Technology (DArT) in evolutionary studies of non-model organisms. DArT is a hybridization-based genotyping method that uses microarray technology to identify and type DNA polymorphism. Theoretically applicable to any organism (even one for which no prior genetic data are available), DArT has not yet been explored in exclusively wild sample sets, nor extensively examined in a phylogenetic framework. DArT recovered 1349 markers of largely low copy-number loci in two lineages of seed-free land plants: the diploid fern Asplenium viride and the haploid moss Garovaglia elegans. Direct sequencing of 148 of these DArT markers identified 30 putative loci including four routinely sequenced for evolutionary studies in plants. Phylogenetic analyses of DArT genotypes reveal phylogeographic and substrate specificity patterns in A. viride, a lack of phylogeographic pattern in Australian G. elegans, and additive variation in hybrid or mixed samples. Conclusions/Significance: These results enable methodological recommendations including procedures for detecting and analysing DArT markers tailored specifically to evolutionary investigations and practical factors informing the decision to use DArT, and raise evolutionary hypotheses concerning substrate specificity and biogeographic patterns. Thus DArT is a demonstrably valuable addition to the set of existing molecular approaches used to infer biological phenomena such as adaptive radiations, population dynamics, hybridization, introgression, ecological differentiation and phylogeography

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

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

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

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:</p

Targeting of Slc25a21 Is Associated with Orofacial Defects and Otitis Media Due to Disrupted Expression of a Neighbouring Gene

<div><p>Homozygosity for <i>Slc25a21^{tm1a(KOMP)Wtsi}</i> 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 <i>SLC25A21</i> may be involved in the disease 2-oxoadipate acidaemia. Unexpectedly, no 2-oxoadipate acidaemia-like symptoms were observed in animals homozygous for <i>Slc25a21^{tm1a(KOMP)Wtsi}</i> despite confirmation that this allele reduces <i>Slc25a21</i> expression by 71.3%. To study the complete knockout, an allelic series was generated using the <i>loxP</i> and <i>FRT</i> sites typical of a Knockout Mouse Project allele. After removal of the critical exon and neomycin selection cassette, <i>Slc25a21</i> knockout mice homozygous for the <i>Slc25a21^{tm1b(KOMP)Wtsi}</i> and <i>Slc25a21^{tm1d(KOMP)Wtsi}</i> alleles were phenotypically indistinguishable from wild-type. This led us to explore the genomic environment of <i>Slc25a21</i> and to discover that expression of <i>Pax9</i>, located 3′ of the target gene, was reduced in homozygous <i>Slc25a21^{tm1a(KOMP)Wtsi}</i> mice. We hypothesize that the presence of the selection cassette is the cause of the down regulation of <i>Pax9</i> observed. The phenotypes we observed in homozygous <i>Slc25a21^{tm1a(KOMP)Wtsi}</i> mice were broadly consistent with a hypomorphic <i>Pax9</i> allele with the exception of otitis media and hearing impairment which may be a novel consequence of <i>Pax9</i> 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.</p></div

Plant peroxisomal ABC transporters: flexible and unusual

ABC transporters of subfamily D mediate import of substrates for β-oxidation into peroxisomes. Whilst mammals possess three peroxisomal ABCD proteins which homodimerise to form transporters with distinct substrate specificities, Baker’s yeast has a single transporter formed by heterodimerisation, which imports long-chain fatty acyl CoAs. Plants have a single-fused heterodimer transporter that exhibits broad substrate specificity, reflecting the wide range of β-oxidation substrates processed by plants. The fusion appears to have occurred early in the evolution of land plants and was followed by an early duplication event in the monocot lineage. Plant ABCD proteins function in all stages of the life cycle and their physiological roles reflect the ability to transport diverse substrates including saturated and unsaturated fatty acids and aromatic compounds such as precursors of hormones and secondary metabolites. Recent work suggests that transport of CoA substrates involves their cleavage and re-esterification within the peroxisome, thus interaction with appropriate acyl adenylate-activating enzymes potentially provides a mechanism for regulating entry of different substrates into β-oxidation. The mechanism of ABCD transporter targeting is broadly conserved across kingdoms but evidence suggests the regulation of protein turnover differs