James Bussell

Genome-wide Generation and Systematic Phenotyping of Knockout Mice Reveals New Roles for Many Genes

Summary 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 We found that hitherto unpublished genes were as likely to reveal phenotypes as known genes, suggesting that novel genes represent a rich resource for investigating the molecular basis of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

Experimental and husbandry procedures as potential modifiers of the results of phenotyping tests.

To maximize the sensitivity of detecting affects of genetic variants in mice, variables have been minimized through the use of inbred mouse lines, by eliminating infectious organisms and controlling environmental variables. However, the impact of standard animal husbandry and experimental procedures on the validity of experimental data is under appreciated. In this study we monitored the impact of these procedures by using parameters that reflect stress and physiological responses to it. Short-term measures included telemetered heart rate and systolic arterial pressure, core body temperature and blood glucose, while longer-term parameters were assessed such as body weight. Male and female C57BL6/NTac mice were subjected to a range of stressors with different perceived severities ranging from repeated blood glucose and core temperature measurement procedures, intra-peritoneal injection and overnight fasting to cage transport and cage changing. Our studies reveal that common husbandry and experimental procedures significantly influence mouse physiology and behaviour. Systolic arterial pressure, heart rate, locomotor activity, core temperature and blood glucose were elevated in response to a range of experimental procedures. Differences between sexes were evident, female mice displayed more sustained cardiovascular responses and locomotor activity than male mice. These results have important implications for the design and implementation of multiple component experiments where the lasting effects of stress from previous tests may modify the outcomes of subsequent ones.

Establishment of mouse expanded potential stem cells

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.

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

Homozygosity for Slc25a21tm1a(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 Slc25a21tm1a(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 Slc25a21tm1b(KOMP)Wtsi and Slc25a21tm1d(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 Slc25a21tm1a(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 Slc25a21tm1a(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.

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

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.

Evaluating and Optimizing Fish Health and Welfare During Experimental Procedures

Many facilities house fish in separate static containers post-procedure, for example, while awaiting genotyping results. This ensures fish can be easily identified, but it does not allow for provision of continuous filtered water or diet. At the Wellcome Trust Sanger Institute, concern over the housing conditions led to the development of an individual housing system (GeneS) enabling feeding and water filtration. Trials to compare the water quality measures between the various systems found that fish housed in static containers experienced rapid deterioration in water quality. By day 1, measures of ammonia were outside the Institutes prescribed values and continued to rise until it was 25-fold higher than recommended levels. Nitrite levels were also outside recommended levels for all fish by day 9 and were twofold higher by the end of the trial. The water quality measures for tanks held on the recirculating system were stable even though food was provided. These results indicate that for housing zebrafish, running water or appropriately timed water changes are a critical component to ensure that the ethical obligations are met.

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

ABSTRACT 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. Editors Choice: The report presents 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.

Talking welfare: the importance of a common language

Ontologies describing mouse phenotypes and pathology are well established and becoming more universally used (Smith and Eppig in Mamm Genome 23:653, 2012; Scofield et al. in J Biomed Semant 4:18, 2013). However, the language used to describe and disseminate cage-side observations is less well developed. This article explores the hurdles to unifying a language and terminology, and introduces our initial attempt to do so.

A risk-based approach to reducing exposure of staff to laboratory animal allergens

Within the biomedical research industry, people who work with laboratory animals may be at risk of developing laboratory animal allergy, which can lead to occupational asthma. Under UK and EU laws, employers must prevent or adequately control exposure to any hazardous substance, which includes animal allergens, so far as reasonably practicable, for the protection of all people on the premises. This can be achieved in part by reviewing the risk of allergen exposure in specific areas of a facility and implementing appropriate infrastructure, environmental and performance controls to minimize that risk. The authors describe the approach used at their institution to stratify risk of allergen exposure in various areas of the animal facility and to implement appropriate controls. They also discuss their use of a monitoring program to evaluate allergen concentrations in low- and high-risk areas of the animal facility and explain how the monitoring results can be applied to determine which controls are needed to minimize risk of exposure and to provide a safe working environment.

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

We report an albino C57BL/6N mouse strain carrying a spontaneous mutation in the tyrosinase gene (C57BL/6N‐TyrcWTSI). 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. genesis 51:523–528.