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 We found that hitherto unpublished genes were as likely to reveal phenotypes as known genes, suggesting that novel genes represent a rich resource for investigating the molecular basis of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

Video-assisted cardiopulmonary resuscitation via smartphone improves quality of resuscitation: A randomised controlled simulation trial.

BACKGROUND: Despite intensive research, cardiac arrest remains a leading cause of death. It is of paramount importance to undertake every possible effort to increase the overall quality of cardiopulmonary resuscitation (CPR) and improve patient outcome. CPR initiated by a bystander is one of the key factors in survival of such an incident. Telephone-assisted CPR (T-CPR) has proved to be an effective measure in improving layperson resuscitation. OBJECTIVE: We hypothesised that adding video-telephony to the emergency call (video-CPR, V-CPR) enhances the quality of layperson resuscitation. DESIGN: This randomised controlled simulation trial was performed from July to August 2018. Laypersons were randomly assigned to video-assisted (V-CPR), telephone-assisted (T-CPR) or control (unassisted CPR) groups. Participants were instructed to perform first aid on a mannequin during a simulated cardiac arrest. SETTING: This study was conducted in the Skills Lab of the University Hospital of Cologne. PARTICIPANTS: One hundred and fifty healthy adult volunteers. INTERVENTION: The participants received a smartphone to call emergency services, with Emergency Eye video-call in V-CPR group, and normal telephone functionality in the other groups. T-CPR and V-CPR groups received standardised CPR assistance via phone. MAIN OUTCOME MEASURES: Our primary endpoint was resuscitation quality, quantified by compression frequency and depth, and correct hand position. RESULTS: Mean compression frequency of V-CPR group was 106.4 ±â€Š11.7 min, T-CPR group 98.9 ±â€Š12.3 min (NS), unassisted group 71.6 ±â€Š32.3 min (P < 0.001). Mean compression depth was 55.4 ±â€Š12.3 mm in V-CPR, 52.1 ±â€Š13.3 mm in T-CPR (P < 0.001) and 52.9 ±â€Š15.5 mm in unassisted (P < 0.001). Total percentage of correct chest compressions was significantly higher (P < 0.001) in V-CPR (82.6%), than T-CPR (75.4%) and unassisted (77.3%) groups. CONCLUSION: V-CPR was shown to be superior to unassisted CPR, and was comparable to T-CPR. However, V-CPR leads to a significantly better hand position compared with the other study groups. V-CPR assistance resulted in volunteers performing chest compressions with more accurate compression depth. Despite reaching statistical significance, this may be of little clinical relevance. TRIAL REGISTRATION: ClinicalTrials.gov (Identifier: NCT03527771).

The critical role of histone H2A-deubiquitinase Mysm1 in hematopoiesis and lymphocyte differentiation.

Stem cell differentiation and lineage specification depend on coordinated programs of gene expression, but our knowledge of the chromatin-modifying factors regulating these events remains incomplete. Ubiquitination of histone H2A (H2A-K119u) is a common chromatin modification associated with gene silencing, and controlled by the ubiquitin-ligase polycomb repressor complex 1 (PRC1) and H2A-deubiquitinating enzymes (H2A-DUBs). The roles of H2A-DUBs in mammalian development, stem cells, and hematopoiesis have not been addressed. Here we characterized an H2A-DUB targeted mouse line Mysm1(tm1a/tm1a) and demonstrated defects in BM hematopoiesis, resulting in lymphopenia, anemia, and thrombocytosis. Development of lymphocytes was impaired from the earliest stages of their differentiation, and there was also a depletion of erythroid cells and a defect in erythroid progenitor function. These phenotypes resulted from a cell-intrinsic requirement for Mysm1 in the BM. Importantly, Mysm1(tm1a/tm1a) HSCs were functionally impaired, and this was associated with elevated levels of reactive oxygen species, γH2AX DNA damage marker, and p53 protein in the hematopoietic progenitors. Overall, these data establish a role for Mysm1 in the maintenance of BM stem cell function, in the control of oxidative stress and genetic stability in hematopoietic progenitors, and in the development of lymphoid and erythroid lineages.

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

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.

Do laypersons need App-linked real-time feedback devices for effective resuscitation? - Results of a prospective, randomised simulation trial.

Background: App-linked real-time feedback-devices for cardiopulmonary resuscitation (CPR) aim to improve laypersons' resuscitation quality. Resuscitation guidelines recommend these technologies in training settings. This is the first study comparing resuscitation quality of all App-linked feedback-devices currently on market. Methods: A prospective randomised simulation study was performed. After standardised instructions, participants performed 2-minutes compression-only CPR on a manikin without feedback (baseline). Afterwards, participants performed 4 × 2 min CPR with four different feedback devices in randomised order (CorPatch® Trainer, CPRBAND AIO Training, SimCPR®ProTrainer, Relay Response™) (intervention). CPR metrics (chest compression depth (CD), chest compression rate (CR), percentage of correct CD/CR (%), correct hand position, correct chest recoil, and technical preparation-time) were assessed. Devices data were compared to the baseline group using Wilcoxon testing with IBM SPSS (primary outcome). Differences between devices were analysed with ANOVA testing (secondary outcome). Normally distributed data were described as mean ± standard deviation (SD) and non-normally distributed data as Median [Interquartile range (IQR). CPR self-confidence was measured by means of questionnaire before and after feedback devices' use. Comparison was performed by students t-test. Results: Forty participants were involved. SimCPR®ProTrainer was the only device, which resulted in guideline-compliant chest compressions (Mean ± SD:5.37 ± 0.76) with improved chest compression depth (p < 0.001), and percentage of correct chest compression depth (p < 0.001) compared to unassisted CPR (baseline). CorPatch® Trainer as the only device with audio-visual recoil instructions resulted in improved chest recoil (Mean ± SD:72.25 ± 24.89) compared to baseline (Mean ± SD:49.00 ± 42.20; p < 0.01), while the other three devices resulted in significantly lower chest recoil rates (CPRBAND AIO Training: 37.03 ± 39.90; p < 0.01, SimCPR®ProTrainer: Mean ± SD:39.88 ± 36.50; p = 0.03, Relay Response™: Mean ± SD:36.88 ± 37.73; p = 0.02). CPR quality when using the different feedback devices differ in chest compression depth (p = 0.02), chest compression rate (p < 0.001), percentage of correct chest compression depth/rate (p = 0.03/p = 0.04), and technical preparation-time (p < 0.001). Feedback-devices' use increased participant's CPR self-confidence (p < 0.001). Conclusion: Although, CPR feedback devices show improved CPR performance in layperson in some metrics, none of the tested CPR feedback devices supported layperson in overall adequate CPR performance. More and better technical functionality is necessary, to fully utilise the potential of CPR feedback devices and to prevent a worsening of CPR performance when layperson use this technology.

Alternative techniques for cardiopulmonary resuscitation in extreme environments - A scoping review.

Background: Cardiopulmonary resuscitation (CPR) is essential for saving lives during cardiac arrest, but performing CPR in extreme environments poses unique challenges. In scenarios ranging from hypogravity or microgravity to confined spaces like aeroplanes and underwater scenarios, traditional CPR techniques may be inadequate. This scoping review aims to identify alternative chest compression techniques, synthesise current knowledge, and pinpoint research gaps in resuscitation for cardiac arrest in extreme conditions. Methods: PubMed and the Cochrane Register of Controlled Trials as well as the website of ResearchGate was searched to identify relevant literature. Studies were eligible for inclusion if they evaluated alternative chest compression techniques, including manual or mixed CPR approaches, whilst assessing feasibility and effectiveness based on compression depth, rate, and/or impact on rescuer effort. Results: The database search yielded 9499 references. After screening 26 studies covering 6 different extreme environments were included (hypogravity: 2; microgravity: 9, helicopter: 1, aeroplane: 1, confined space: 11; avalanche: 2). 13 alternative chest compression techniques were identified, all of which tested using manikins to simulate cardiac arrest scenarios. Conclusion: To address the unique challenges in extreme environments, novel CPR techniques are emerging. However, evidence supporting their effectiveness remains limited.

MouseBook: an integrated portal of mouse resources.

The MouseBook (http://www.mousebook.org) databases and web portal provide access to information about mutant mouse lines held as live or cryopreserved stocks at MRC Harwell. The MouseBook portal integrates curated information from the MRC Harwell stock resource, and other Harwell databases, with information from external data resources to provide value-added information above and beyond what is available through other routes such as International Mouse Stain Resource (IMSR). MouseBook can be searched either using an intuitive Google style free text search or using the Mammalian Phenotype (MP) ontology tree structure. Text searches can be on gene, allele, strain identifier (e.g. MGI ID) or phenotype term and are assisted by automatic recognition of term types and autocompletion of gene and allele names covered by the database. Results are returned in a tabbed format providing categorized results identified from each of the catalogs in MouseBook. Individual result lines from each catalog include information on gene, allele, chromosomal location and phenotype, and provide a simple click-through link to further information as well as ordering the strain. The infrastructure underlying MouseBook has been designed to be extensible, allowing additional data sources to be added and enabling other sites to make their data directly available through MouseBook.

EuroPhenome: a repository for high-throughput mouse phenotyping data.

The broad aim of biomedical science in the postgenomic era is to link genomic and phenotype information to allow deeper understanding of the processes leading from genomic changes to altered phenotype and disease. The EuroPhenome project (http://www.EuroPhenome.org) is a comprehensive resource for raw and annotated high-throughput phenotyping data arising from projects such as EUMODIC. EUMODIC is gathering data from the EMPReSSslim pipeline (http://www.empress.har.mrc.ac.uk/) which is performed on inbred mouse strains and knock-out lines arising from the EUCOMM project. The EuroPhenome interface allows the user to access the data via the phenotype or genotype. It also allows the user to access the data in a variety of ways, including graphical display, statistical analysis and access to the raw data via web services. The raw phenotyping data captured in EuroPhenome is annotated by an annotation pipeline which automatically identifies statistically different mutants from the appropriate baseline and assigns ontology terms for that specific test. Mutant phenotypes can be quickly identified using two EuroPhenome tools: PhenoMap, a graphical representation of statistically relevant phenotypes, and mining for a mutant using ontology terms. To assist with data definition and cross-database comparisons, phenotype data is annotated using combinations of terms from biological ontologies.

Optimising experimental design for high-throughput phenotyping in mice: a case study.

To further the functional annotation of the mammalian genome, the Sanger Mouse Genetics Programme aims to generate and characterise knockout mice in a high-throughput manner. Annually, approximately 200 lines of knockout mice will be characterised using a standardised battery of phenotyping tests covering key disease indications ranging from obesity to sensory acuity. From these findings secondary centres will select putative mutants of interest for more in-depth, confirmatory experiments. Optimising experimental design and data analysis is essential to maximise output using the resources with greatest efficiency, thereby attaining our biological objective of understanding the role of genes in normal development and disease. This study uses the example of the noninvasive blood pressure test to demonstrate how statistical investigation is important for generating meaningful, reliable results and assessing the design for the defined research objectives. The analysis adjusts for the multiple-testing problem by applying the false discovery rate, which controls the number of false calls within those highlighted as significant. A variance analysis finds that the variation between mice dominates this assay. These variance measures were used to examine the interplay between days, readings, and number of mice on power, the ability to detect change. If an experiment is underpowered, we cannot conclude whether failure to detect a biological difference arises from low power or lack of a distinct phenotype, hence the mice are subjected to testing without gain. Consequently, in confirmatory studies, a power analysis along with the 3Rs can provide justification to increase the number of mice used.

F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses.

A useful approach for exploring gene function involves generating mutant mice from genetically modified embryonic stem (ES) cells. Recent advances in genetic engineering of ES cells have shifted the bottleneck in this process to the generation of mice. Conventional injections of ES cells into blastocyst hosts produce F0 generation chimeras that are only partially derived from ES cells, requiring additional breeding to obtain mutant mice that can be phenotyped. The tetraploid complementation approach directly yields mice that are almost entirely derived from ES cells, but it is inefficient, works only with certain hybrid ES cell lines and suffers from nonspecific lethality and abnormalities, complicating phenotypic analyses. Here we show that laser-assisted injection of either inbred or hybrid ES cells into eight cell-stage embryos efficiently yields F0 generation mice that are fully ES cell-derived and healthy, exhibit 100% germline transmission and allow immediate phenotypic analysis, greatly accelerating gene function assignment.

Rescue under ongoing CPR from an upper floor: evaluation of three different evacuation routes and mechanical and manual chest compressions: a manikin trial.

BACKGROUND: If transport under ongoing cardiopulmonary resuscitation (CPR) from an upper floor is indicated, the ideal CPR-method and evacuation route is unknown hitherto. We aimed to elaborate a strategy for evacuation of patients under ongoing CPR from an upper floor, comparing three different evacuation routes and manual and mechanical chest compressions. METHODS: A CPR-training manikin recording CPR-quality was placed on the fifth floor and was evacuated to an ambulance via lift, turntable ladder, or staircase. Chest compressions were performed manually or with a mechanical CPR-device. Efficiency endpoints were compression depth and frequency, sufficiency of chest release, compared with European Resuscitation Council (ERC) Guidelines, and duration of the evacuation. Adverse outcomes were disconnection/dislocation of devices and hazards/accidents to the personnel. RESULTS: For all evacuation routes, compression depth and frequency were significantly more compliant with ERC-guidelines under mechanical CPR. Manual CPR was associated with considerable deviations from correct compression depth and frequency. Chest release only slightly differed between groups. Evacuation via lift under mechanical CPR was fastest and evacuation via turntable ladder under manual CPR was slowest. No device disconnections or accidents occurred, but hazard to personnel was perceived during evacuation via ladder under manual CPR. CONCLUSIONS: In this study, a mechanical CPR-device proved to deliver better CPR-quality during evacuation from an upper floor. If a lift accessible with a stretcher is available, this route should be preferred, regardless of manual or mechanical CPR. Turntable ladders can only be meaningfully used with mechanical CPR, otherwise CPR-quality is poor and hazard to the personnel is increased. Not all evacuation routes may be useable in a specific real-life scenario. TRIAL REGISTRATION: German Clinical Trials Registry, www.drks.de, registration number DRKS00012885, registration date 17.08.2017.

A divergent pattern of sensory axonal projections is rendered convergent by second-order neurons in the accessory olfactory bulb.

The mammalian vomeronasal system is specialized in pheromone detection. The neural circuitry of the accessory olfactory bulb (AOB) provides an anatomical substrate for the coding of pheromone information. Here, we describe the axonal projection pattern of vomeronasal sensory neurons to the AOB and the dendritic connectivity pattern of second-order neurons. Genetically traced sensory neurons expressing a given gene of the V2R class of vomeronasal receptors project their axons to six to ten glomeruli distributed in globally conserved areas of the AOB, a theme similar to V1R-expressing neurons. Surprisingly, second-order neurons tend to project their dendrites to glomeruli innervated by axons of sensory neurons expressing the same V1R or the same V2R gene. Convergence of receptor type information in the olfactory bulb may represent a common design in olfactory systems.

Novel modulation of neuronal nicotinic acetylcholine receptors by association with the endogenous prototoxin lynx1.

We previously identified lynx1 as a neuronal membrane molecule related to snake alpha-neurotoxins able to modulate nAChRs. Here, we show that lynx1 colocalizes with nAChRs on CNS neurons and physically associates with nAChRs. Single-channel recordings show that lynx1 promotes the largest of three current amplitudes elicited by ACh through alpha(4)beta(2) nAChRs and that lynx1 enhances desensitization. Macroscopic recordings quantify the enhancement of desensitization onset by lynx1 and further show that it slows recovery from desensitization and increases the EC(50). These experiments establish that direct interaction of lynx1 with nAChRs can result in a novel type of functional modulation and suggest that prototoxins may play important roles in vivo by modulating functional properties of their cognate CNS receptors.

Peripheral myelin protein 22 is in complex with alpha6beta4 integrin, and its absence alters the Schwann cell basal lamina.

Peripheral myelin protein 22 (PMP22) is a tetraspan membrane glycoprotein, the misexpression of which is associated with hereditary demyelinating neuropathies. Myelinating Schwann cells (SCs) produce the highest levels of PMP22, yet the function of the protein in peripheral nerve biology is unresolved. To investigate the potential roles of PMP22, we engineered a novel knock-out (-/-) mouse line by replacing the first two coding exons of pmp22 with the lacZ reporter. PMP22-deficient mice show strong beta-galactosidase reactivity in peripheral nerves, cartilage, intestines, and lungs, whereas phenotypically they display the characteristics of tomaculous neuropathy. In the absence of PMP22, myelination of peripheral nerves is delayed, and numerous axon-SC profiles show loose basal lamina, suggesting altered interactions of the glial cells with the extracellular matrix. The levels of beta4 integrin, a molecule involved in the linkage between SCs and the basal lamina, are severely reduced in nerves of PMP22-deficient mice. During early stages of myelination, PMP22 and beta4 integrin are coexpressed at the cell surface and can be coimmunoprecipitated together with laminin and alpha6 integrin. In agreement, in clone A colonic carcinoma cells, epitope-tagged PMP22 forms a complex with beta4 integrin. Together, these data indicate that PMP22 is a binding partner in the integrin/laminin complex and is involved in mediating the interaction of SCs with the extracellular environment.

High-throughput engineering of the mouse genome coupled with high-resolution expression analysis.

One of the most effective approaches for determining gene function involves engineering mice with mutations or deletions in endogenous genes of interest. Historically, this approach has been limited by the difficulty and time required to generate such mice. We describe the development of a high-throughput and largely automated process, termed VelociGene, that uses targeting vectors based on bacterial artificial chromosomes (BACs). VelociGene permits genetic alteration with nucleotide precision, is not limited by the size of desired deletions, does not depend on isogenicity or on positive-negative selection, and can precisely replace the gene of interest with a reporter that allows for high-resolution localization of target-gene expression. We describe custom genetic alterations for hundreds of genes, corresponding to about 0.5-1.0% of the entire genome. We also provide dozens of informative expression patterns involving cells in the nervous system, immune system, vasculature, skeleton, fat and other tissues.

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.

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

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.

Mice genetically deficient in neuromedin U receptor 2, but not neuromedin U receptor 1, have impaired nociceptive responses.

Neuromedin U (NMU) has recently been reported to have a role in nociception and inflammation. To clarify the function of the two known NMU receptors, NMU receptor 1 (NMUR1) and NMU receptor 2 (NMUR2), during nociception and inflammation in vivo, we generated mice in which the genes for each receptor were independently deleted. Compared to wild type littermates, mice deficient in NMUR2 showed a reduced thermal nociceptive response in the hot plate, but not in the tail flick, test. In addition, the NMUR2 mutant mice showed a reduced behavioral response and a marked reduction in thermal hyperalgesia following capsaicin injection. NMUR2-deficient mice also showed an impaired pain response during the chronic, but not acute, phase of the formalin test. In contrast, NMUR1-deficient mice did not show any nociceptive differences compared to their wild type littermates in any of the behavioral tests used. We observed the same magnitude of inflammation in both lines of NMU receptor mutant mice compared to their wild type littermates after injection with complete Freund's adjuvant (CFA), suggesting no requirement for either receptor in this response. Thus, the pro-nociceptive effects of NMU in mice appear to be mediated through NMUR2, not NMUR1.

Mouse Phenotype Database Integration Consortium: integration [corrected] of mouse phenome data resources.

Understanding the functions encoded in the mouse genome will be central to an understanding of the genetic basis of human disease. To achieve this it will be essential to be able to characterize the phenotypic consequences of variation and alterations in individual genes. Data on the phenotypes of mouse strains are currently held in a number of different forms (detailed descriptions of mouse lines, first-line phenotyping data on novel mutations, data on the normal features of inbred lines) at many sites worldwide. For the most efficient use of these data sets, we have initiated a process to develop standards for the description of phenotypes (using ontologies) and file formats for the description of phenotyping protocols and phenotype data sets. This process is ongoing and needs to be supported by the wider mouse genetics and phenotyping communities to succeed. We invite interested parties to contact us as we develop this process further.