Breeding Of Mice Research Articles

Normal male fertility in a mouse model of KPNA2 deficiency.

The nuclear transport of proteins is mediated by karyopherins and has been implicated to be crucial for germ cell and embryonic development. Deletion of distinct members of the karyopherin alpha family has been shown to cause male and female infertility in mice. Using a genetrap approach, we established mice deficient for KPNA2 (KPNA2 KO) and investigated the role of this protein in male germ cell development and fertility. Breeding of male KPNA2 KO mice leads to healthy offsprings in all cases albeit the absence of KPNA2 resulted in a reduction in sperm number by 60%. Analyses of the KPNA2 expression in wild-type mice revealed a strong KPNA2 presence in meiotic germ cells of all stages while a rapid decline is found in round spermatids. The high KPNA2 expression throughout all meiotic stages of sperm development suggests a possible function of KPNA2 during this phase, hence in its absence the spermatogenesis is not completely blocked. In KPNA2 KO mice, a higher portion of sperms presented with morphological abnormalities in the head and neck region, but a severe spermiogenesis defect was not found. Thus, we conclude that the function of KPNA2 in round spermatids is dispensable, as our mice do not show any signs of infertility. Our data provide evidence that KPNA2 is not crucial for male germ cell development and fertility.

Proper micro-environment alleviates mortality in laboratory mouse breeding induced by litter overlap and older dams

The ongoing worldwide effort to reduce animal numbers in research often omits the issue of pre-weaning mortality in mouse breeding. A conservative estimate of 20% mortality would mean approximately 1.1 M mice die annually in the EU before scientific use. We hypothesize that pre-weaning mortality in laboratory mouse breeding is associated with cage social and macro/micro-environment conditions. Here we count pups from 509 C57BL/6J litters daily for accurate detection of mortality, and monitor cage micro-environment for 172 C57BL/6J litters. Probability of pups to die increases with the increase in dam age, number and age of older pups in the cage (of overlapped/cohabitating litters), and in small (<6 pups) and large (>11 pups) focal litters. Higher temperatures (>23.6 °C) and nest scores (>3.75) compensate for some of the socially-associated risks for pup death. These findings can be implemented in strategies for reducing pre-weaning mouse mortality, a more welfare-friendly and sustainable approach for science.

Adult Offspring of Deer Mouse Breeding Pairs Selected for Normal and Compulsive-Like Large Nesting Expression Invariably Show the Same Behavior Without Prior In-Breeding.

Obsessive-compulsive disorder is a neuropsychiatric condition with notable genetic involvement. Against this background, laboratory-housed deer mice of both sexes varyingly present with excessive and persistent large nesting behavior (LNB), which has been validated for its resemblance of clinical compulsivity. Although LNB differs from normal nesting behavior (NNB) on both a biological and cognitive level, it is unknown to what extent the expression of LNB and NNB is related to familial background. Here, we randomly selected 14 NNB- and 14 LNB-expressing mice (equally distributed between sexes) to constitute 7 breeding pairs of each phenotype. Pairs were allowed to breed two successive generations of offspring, which were raised until adulthood (12 weeks) and assessed for nesting expression. Remarkably, our findings show that offspring from LNB-expressing pairs build significantly larger nests compared to offspring from NNB-expressing pairs and the nesting expression of the offspring of each breeding pair, irrespective of parental phenotype or litter, is family specific. Collectively, the results of this investigation indicate that LNB can be explored for its potential to shed light on heritable neurocognitive mechanisms that may underlie the expression of specific persistent behavioral phenotypes.

Methods for Decreasing Preweaning Mortality in a Fragile Mouse Model of Hypomorphic Collagen VII Deficiency.

Preweaning mortality is a widespread problem in laboratory mouse breeding, particularly in the case of fragile mouse models. While numerous studies explore alternative care methods to increase the survivability of common mouse strains, there remains a paucity of research into the care of mice with fragile health conditions that result from induced or natural genetic mutations. In this study, standard husbandry practices were enhanced by the addition of a softened diet, a nutritionally fortified dietary supplement, soft bedding, gentle handling techniques, decreased handling, lengthened weaning age, and dam productivity tracking. This alternative care plan was shown to increase the survival of a fragile recessive dystrophic epidermolysis bullosa mouse model, and some aspects could be used in developing a care plan for other fragile mouse strains.

Effect of Cage Change Frequency on Perinatal Mortality in C57BL/6J Mice.

Perinatal mortality is a common problem in mouse breeding colonies. Few studies have examined the influence of environmental changes on mouse pup survival. In this study, monogamous breeding cages of C57BL/6J mice were set up and randomized into 3 cage change groups: 1) cage change at 8 d after parturition, 2) cage change at 3 d after parturition, or 3) cage change at 3 d after parturition with the addition of a polycarbonate hut in the cage. Pairs were bred to produce a minimum of 4 litters. Pup survival to weaning relative to experimental cage change date, and survival rates after cage change were evaluated. The results revealed no significant differences between experimental groups. The majority of pup loss occurred within the first 24 h after birth for those pups that were alive at birth. Overall, the postpartum day of cage change did not affect the perinatal survival of mouse pups.

RAAV8-PC Gene Therapy for Severe Protein C Deficiency Using a Novel Murine Model

Introduction Protein C (PC) is a vitamin K-dependent protein, synthesized in the liver. The PROC gene is located on chromosome 2 at position 2q13-14 and encodes 9 exons. Severe protein C deficiency (SPCD) is a rare devastating autosomal recessive disease that presents at birth with purpura fulminans (PF) and is associated with high rates of morbidity and mortality. As current management options are limited and burdensome, there is a large unmet need for new treatments including curative therapies. Because small increases in PC may significantly ameliorate the clinical presentation of SPCD and potentially prevent PF, we hypothesize that SPCD could be treated by PC gene therapy (GT). Aim To establish a murine model enabling the survival of SPCD mice and to evaluate PC expression after recombinant adeno-associated viral vector- PC (rAAV8-PC) gene therapy. Methods The study was conducted with C57Bl/6 PC heterozygous mice (a kind gift of Prof. Weiler, [Wisconsin]), severe hemophilia A (HA) mice and wild type C57Bl/6 mice. Genotyping was performed by PCR using appropriate primers for murine PROC and F8 genes. rAAV8-PC was produced by transfecting HEK293 cells with PX-680 as rAAV helper, AAV8 and PC plasmid with an alpha-1 antitrypsin promotor. Extraction and purification were performed using a commercial kit. Viral concentration was determined by a designated commercial qPCR assay. rAAV8 - PC (10 12 /kg Vg of AAV8-PC) was injected via the tail vein to 5-8 weeks old mice. Blood samples were collected from the facial vein into 1.5 ml tubes containing 0.109M sodium citrate at blood: anticoagulant ratio of 1:1 (vol/vol). PC plasma levels were measured prior and following rAAV8-PC infusion using a commercial ELISA assay for PC antigen. Thrombin generation was measured in plasma samples using a modification of the calibrated automated thrombinoscope assay. Results As expected, breeding of heterozygous PC mice yielded only 3/200 homozygous SPCD mice (PC levels= 0), that survived for less than 24 hours. Thus, GT for heterozygous PC mice was considered; however, PC plasma levels in heterozygous PC mice were not substantially different from those in WT mice (median levels in heterozygous PC and WT mice: 216 ng/ml vs. 190 ng/ml, respectively), which precluded this option. Hence, with the aim of increasing the survival rate and duration of homozygous SPCD mice, we decided to rebalance the severe thrombotic disorder of SPCD, by induction of a genetically engineered combination with the severe coagulopathy of Hemophilia. We established an alternative SPCD murine model by breeding severe HA mice with heterozygous PC mice. This approach yielded homozygous SPCD mice with severe HA (PROC-/F8-), exhibiting a significantly prolonged survival compared to homozygous SPCD mice with normal F8 levels (over 90% survived over 6 weeks). Genotyping and ELISA of PC levels confirmed PC homozygosity (null mutation) as well as severe HA. Nineteen PROC-/F8- mice were injected with rAAV8-PC into the tail vein and followed for at least 12 weeks. Nine mice died during blood sampling at different time points and were excluded from further analysis. Individual responses to PC- GT were noted among the remaining 10 mice, reflected by non-uniform PC expression measured by ELISA (initial PC increase, time to peak and PC peak plasma levels). An increase in PC level was noted at week 2-5 post vector infusion, reaching PC peak level between week 2-10 post GT. Table 1 summarizes the individual patterns of 10 PROC-/F8-mice that underwent GT. The functional activity of PC in the murine plasma following GT was supported by thrombin generation assays. Analyses performed in PROC-/F8-mice before and after GT demonstrated endogenous thrombin potential (ETP) and peak hight decrease following GT, as compared to untreated PROC-/F8-mice. Conclusion We present here a novel murine model of combined PC and factor VIII deficiency that supports the survival of SPCD mice and enables PC gene therapy studies. Our results provide the first proof of concept that infusion of rAAV8-PC results in increased PC plasma levels in mice, and may serve as a basis for future studies investigating GT for SPCD. The concept of modulating a severe genetic prothrombotic disorder, which is nearly incompatible with life, by genetic combination with severe coagulopathy may offer a valuable tool enabling investigation and potential development of treatment modalities for the prothrombotic disorder.

Repopulation of autophagy-deficient stromal cells with autophagy-intact cells after repeated breeding in uterine mesenchyme-specific Atg7 knockout mice.

Autophagy is highly active in ovariectomized mice experiencing hormone deprivation, especially in the uterine mesenchyme. Autophagy is responsible for the turnover of vasoactive factors in the uterus, which was demonstrated in anti-Müllerian hormone receptor type 2 receptor (Amhr2)-Cre-driven autophagy-related gene 7 (Atg7) knockout (Amhr-Cre/Atg7f/f mice). In that study, we uncovered a striking difference in the amount of sequestosome 1 (SQSTM1) accumulation between virgin mice and breeder mice with the same genotype. Herein, we aimed to determine whether repeated breeding changed the composition of mesenchymal cell populations in the uterine stroma. All female mice used in this study were of the same genotype. Atg7 was deleted by Amhr2 promoter-driven Cre recombinase in the uterine stroma and myometrium, except for a triangular stromal region on the mesometrial side. Amhr-Cre/Atg7f/f female mice were divided into two groups: virgin mice with no mating history and aged between 11 and 12 months, and breeder mice with at least 6-month breeding cycles with multiple pregnancies and aged around 12 months. The uteri were used for Western blotting and immunofluorescence staining. SQSTM1 accumulation, representing Atg7 deletion and halted autophagy, was much higher in virgin mice than in breeders. Breeders showed reduced accumulation of several vasoconstrictive factors, which are potential autophagy targets, in the uterus, suggesting that the uterine stroma was repopulated with autophagy-intact cells during repeated pregnancies. Multiple pregnancies seem to have improved the uterine environment by replacing autophagy-deficient cells with autophagy-intact cells, providing evidence of cell mixing.

Pathologic copulatory lock in a genetically engineered laboratory mouse breeding pair.

A breeding pair of genetically engineered laboratory mice (Mus musculus) presented in apparent copulatory lock (coital tie). After anesthetizing the animals, gentle traction was used to separate the pair at which point a vaginal prolapse was detected and the penis was covered with black, firm, dry crusts and noted to have a solid pale, tan, firm cylindrical mass adhering to its glans. The vaginal prolapse was reduced and the female was returned to its cage. The male mouse had a severely distended bladder which could not be expressed and was euthanized. Histopathologic examination of the distal two-thirds of the penis revealed diffuse, acute coagulative necrosis. The mass adhered to the distal penis was a homogenous granular eosinophilic material consistent with a copulatory plug. While copulatory plugs and locks have been described in some rodent species, they have not been reported in laboratory mice. While the cause of the adherence of the plug to the penis could not be determined, we hypothesize that its adherence to both the penis and the vagina led to the lock and subsequently to ischemic necrosis of the distal penis.

The Type 7b Secretion System of S. aureus and Its Role in Colonization and Systemic Infection.

Staphylococcus aureus bears a type 7b secretion system (T7bSS) that assembles in the bacterial envelope to promote the secretion of WXG-like proteins and toxic effectors bearing LXG domains. Cognate immunity proteins bind cytosolic effectors to mute their toxicity prior to secretion. T7b-secreted factors have been associated with the pathogenesis of staphylococcal disease and intraspecies competition. We identified earlier strain WU1, an S. aureus ST88 isolate that caused outbreaks of skin and soft tissue infections in mouse breeding facilities. WU1 was also found to persistently colonize the nasopharynx of animals, suggesting a strong host adaptation. In this manner, WU1 colonization and infectivity in mice resembles that of methicillin-sensitive and -resistant S. aureus strains in humans, where nasal carriage is a major risk factor for invasive infections. Here, animals were colonized with wild-type or T7-deficient WU1 strains or combinations thereof. Absence of the T7bSS did not affect colonization in the nasopharynx of animals, and although fluctuations were observed in weekly samplings, the wild-type strain did not replace the T7-deficient strain in cocolonization experiments. Bloodstream infection with a T7b-deficient strain resulted in enhanced survival and reduced bacterial loads and abscesses in soft tissues compared to infection with wild-type WU1. Together, experiments using a mouse-adapted strain suggest that the T7bSS of S. aureus is an important contributor to the pathogenesis of invasive disease.

A45 FIBER-FREE DIET REDUCES BACTEROIDES ABUNDANCE AND PREVENTS MUCUS DEGRADATION IN MICE COLONIZED WITH MICROBIOTA FROM PATIENTS WITH GENERALIZED ANXIETY DISORDER

Abstract Background Generalized anxiety disorder (GAD) is a debilitating condition with a lifetime prevalence of 4-7% worldwide. We have previously found that compared to healthy controls, GAD patients had lower reported fiber intake, increased gastrointestinal symptoms; and enrichment of Bacteroides genus as well as carbohydrate metabolism pathways (as determined by PICRUSt2, correlated to Bacteroides abundance). Bacteroides are known for its ability to degrade a wide variety of host polysaccharides, such as the intestinal mucus, which could lead to local and systemic inflammation. In this regard, GAD patients had higher C-reactive protein (CRP) compared to healthy controls (p=0.049). Purpose To investigate whether a fiber-free diet could decrease Bacteroides abundance and prevent damage of the mucus layer reducing anxiety-behavior in mice with GAD microbiota. Method Two germ-free NIH Swiss mouse breeding pairs were colonized with GAD microbiota using patients’ stool samples and kept on either fiber-free or 10 % inulin (fiber) diet. Offspring were weaned at week 3 and psychometric tests were performed at 10 weeks of age. After sacrifice, samples for histology (mucus layer thickness determination), blood (CRP ELISA determination) and stool (Illumina 16S rRNA gene sequencing) were collected. The microbiota data was analyzed following the pipelines of dada2 and by mean comparisons, correlation, AncomBC using R software (v.1.2.1335). Multiple comparison results were corrected allowing 5% of FDR. Result(s) Beta diversity analysis showed that parent and offspring’s (n=24 fiber-supplemented and n=14 fiber-free groups) microbiota was similar to the GAD donor. The most differentially abundant bacterial taxon was Bacteroides uniformis, which was decreased in the fiber-free group (p.adj= 0.003). Furthermore, fiber-free diet reduced the overall Bacteroides abundance by half compared to the donor and fiber-supplemented mice group. This led to a thickening (p.adj=0.027) of the mucus layer, increasing from 30 µm (fiber-supplemented diet) to 60 µm in the fiber-free diet group. B. uniformis was negatively correlated to the mucus layer thickness (R= -0.81; p.adj=0.26), although not statistically significant, likely due to a low n number (n=4). We only found a statistical trend for higher CRP levels and anxiety-like behavior in the fiber-supplemented group. This might be because fiber supplementation has several beneficial effects that can counteract the proposed increase in anxiety-like behavior fromBacteroides. Despite that, we found a significant correlation between B. uniformis and time mice spent in the dark (indicative of anxiety-like behavior) in the light preference test. Conclusion(s) Our data suggests that Bacteroides abundance, specifically Bacteroides uniformis, contributes to the degradation of the mucus layer and potentially triggers low grade gut inflammation and anxiety-like behavior. Please acknowledge all funding agencies by checking the applicable boxes below CIHR Disclosure of Interest None Declared

Effect of Cage Change Frequency on Perinatal Mortality in C57BL/6J Mice

Perinatal mortality is a common problem in mouse breeding colonies. Few studies have examined the influence of environmental changes on mouse pup survival. In this study, monogamous breeding cages of C57BL/6J mice were set up and randomized into 3 cage change groups: 1) cage change at 8 d after parturition, 2) cage change at 3 d after parturition, or 3) cage change at 3 d after parturition with the addition of a polycarbonate hut in the cage. Pairs were bred to produce a minimum of 4 litters. Pup survival to weaning relative to experimental cage change date, and survival rates after cage change were evaluated. The results revealed no significant differences between experimental groups. The majority of pup loss occurred within the first 24 h after birth for those pups that were alive at birth. Overall, the postpartum day of cage change did not affect the perinatal survival of mouse pups.

Single-Cell RNAseq Resolve the Potential Effects of LanCL1 Gene in the Mouse Testis

Infertility affects lots of couples, half of which are caused by male factors. The LanCL1 gene is highly expressed in testis specifically, which might affect the development of sperms. In order to understand the potential functions of the LanCL1 gene in the testis, this study was conducted with constructed transgenic LanCL1 knockout mice. The mouse breeding experiment, semen analysis and single-cell RNAseq of testicular tissue were performed. Results suggested that the LanCL1 gene would significantly influence the reproduction ability and sperm motility of male mice. Single-cell RNAseq also confirmed the high expression of the LanCL1 gene in the spermatocytes and spermatids. Downregulating the LanCL1 gene expression could promote M2 macrophage polarity to maintain testicular homeostasis. Moreover, the LanCL1 gene could affect both the germ cells and stromal cells through various pathways such as the P53 signaling and the PPAR signaling pathway to disturb the normal process of spermatogenesis. However, no effects of the LanCL1 gene in testosterone synthesis and serum testosterone level were shown. Further studies are needed to discuss the mechanisms of the LanCL1 gene in the various cells of the testis independently.

Evaluation of immunogenicity and efficacy of Borrelia burgdorferi derived peptide (PepB) antigen formulated in a protein scaffold in mice against Lyme disease

Lyme disease (LD) is the most prevalent tick-borne disease in both Europe and North America. The development of effective Lyme disease vaccines is complicated by the complex biology of Borrelia species and alterations in the expression of outer surface membranes. In this work, PepB, which is Borrelia burgdorferiBB0172-derived peptide was evaluated in scaffolded formulation as a vaccine candidate in murine model of LD. In brief, four groups of 6-8 weeks old C3H/HeN breed of mice (n=6 per group) were immunized subcutaneously with BBA34: PepB (BP), outer surface protein C (OspC), BBA34 and a naïve control group. In 8 weeks, post-priming blood samples were collected, and specific IgG titers were evaluated by ELISA. After that, A dose of 105 B. burgdorferi/mouse was administered subcutaneously to all animals as a challenge. The mice were euthanized at 4 weeks post-challenge and then blood and tissue samples were collected to evaluate bacterial burden by real time qPCR, and bacterial recovery from tissues. Taken together, and considering both bacterial burden and bacterial recovery, immunization with BP was not able to confer protection against borreliosis in this experiment, and consequently it was not considered as a potential vaccine formulation in subsequent studies. It was concluded, that other alternative antigen platforms and delivery methods might be considered to improve the immunogenicity of the PepB-based vaccine candidate.

Mouse breeding facilities in Argentina: Current state, challenges, and strengths in relation to animal welfare.

The science and technology of laboratory animals has come a long way worldwide, but for reasons related to the development of the countries, this journey started later in some Latin American countries, as is the case of Argentina. Without a specific legal framework to conduct animal experimentation, local strengths to promote animal welfare are based on professionals specifically trained in the care of laboratory animals as well as an extended network of ethics committees that ensures compliance with the ethical principles applied to animal experimentation. Nevertheless, there are no updated reports showing welfare indicators in rodent facilities. Therefore, we conducted a survey on mice breeding facilities enrolled in a national record elaborated by the National Ministry of Science. Questions related to four of the Five Domains Model of Mellor, concerning (1) nutrition, (2) physical environment, (3) health, and (4) behavioral interactions with the environment, other animals, and humans, were included as well as information concerning general aspects of the establishments. Data obtained from 25 mice breeder facilities localized all over the country were summarized, providing for the first time a clear picture of the national situation about the welfare of laboratory mice in these establishments. This data will be essential to design future policy as well as for deciding priorities aiming to improve the welfare of mice bred in Argentinian facilities.

A Journey Into the Unknown: PhD Students in a European Training Network on Age-related Changes in Hematopoiesis Conduct Their Project During a Global Pandemic

A Journey Into the Unknown: PhD Students in a European Training Network on Age-related Changes in Hematopoiesis Conduct Their Project During a Global Pandemic

Impact of Automated Genotyping and Increased Breeding Oversight on Overall Mouse Breeding Colony Productivity.

Mice have become increasingly popular as genetic tools, facilitated by the production of advanced genetically engineered mouse models (GEMMs). GEMMs often require in-house breeding and production by research groups, which can be quite complex depending on the design of the GEMM. Identification of methods to increase the efficiency of breeding practices offers opportunities to optimize and reduce the number of animals bred for research while maintaining similar research output. We investigated the use of commercial automated genotyping and centralized breeding management on overall breeding colony productivity in a colony of multiple GEMM lines. This study involved a three-group study design, where the first group continued their standard breeding practices (group A), the second utilized standard breeding practices but outsourced genotyping in place of inhouse genotyping (group B), and a third group outsourced genotyping and had assistance with routine breeding practices from the laboratory animal care team (group C). Compared to standard practice (group A), groups B and C produced more cages and mice over time, which appeared to be driven primarily by an increase in the number of breeding cages in each colony. Higher numbers of breeders correlated with an increased number of litters and generation of new cages. The increases in colony productivity measures were further enhanced in group C compared to group B. The overall cost associated with producing new animals was lowest in group B, followed by groups A and C. Although, by the end of the study, cost to produce new mice was comparable between all three groups. These data suggest that by optimizing breeding practices and management, fewer animals could be utilized to produce the same amount of progeny and reduce overall animal usage and production.

Investigating audible and ultrasonic noise in modern animal facilities

Background: The environmental housing conditions of laboratory animals are important for both welfare and reliable, reproducible data. Guidelines currently exist for factors such as lighting cycles, temperature, humidity, and noise, however, for the latter the current guidelines may overlook important details. In the case of the most common laboratory species, the mouse, the range of frequencies they can hear is far higher than that of humans. The current guidelines briefly mention that ultrasonic (>20 kHz) frequencies can adversely affect mice, and that the acoustic environment should be checked, though no recommendations are provided relating to acceptable levels of ultrasonic noise. Methods: To investigate the ultrasonic environment in a large mouse breeding facility (the Mary Lyon Centre at MRC Harwell), we compared two systems, the Hottinger Bruel and Kjaer PULSE sound analyser, and an Avisoft Bioacoustics system. Potential noise sources were selected; we used the PULSE system to undertake real-time Fourier analysis of noise up to 100 kHz, and the Avisoft system to record noise up to 125 kHz for later analysis. The microphones from both systems were positioned consistently at the same distance from the source and environmental conditions were identical. In order to investigate our result further, a third system, the AudioMoth (Open Acoustic Devices), was also used for recording. We used DeepSqueak software for most of the recording analysis and, in some cases, we also undertook further spectral analysis using RX8 (iZotope, USA). Results: We found that both systems can detect a range of ultrasonic noise sources, and here discuss the benefits and limitations of each approach. Conclusions: We conclude that measuring the acoustic environment of animal facilities, including ultrasonic frequencies that may adversely affect the animals housed, will contribute to minimising disruption to animal welfare and perturbations in scientific research.

Investigating audible and ultrasonic noise in modern animal facilities.

Background: The environmental housing conditions of laboratory animals are important for both welfare and reliable, reproducible data. Guidelines currently exist for factors such as lighting cycles, temperature, humidity, and noise, however, for the latter the current guidelines may overlook important details. In the case of the most common laboratory species, the mouse, the range of frequencies they can hear is far higher than that of humans. The current guidelines briefly mention that ultrasonic (>20 kHz) frequencies can adversely affect mice, and that the acoustic environment should be checked, though no recommendations are provided relating to acceptable levels of ultrasonic noise. Methods: To investigate the ultrasonic environment in a large mouse breeding facility (the Mary Lyon Centre at MRC Harwell), we compared two systems, the Hottinger Bruel and Kjaer PULSE sound analyser, and an Avisoft Bioacoustics system. Potential noise sources were selected; we used the PULSE system to undertake real-time Fourier analysis of noise up to 100 kHz, and the Avisoft system to record noise up to 125 kHz for later analysis. The microphones from both systems were positioned consistently at the same distance from the source and environmental conditions were identical. In order to investigate our result further, a third system, the AudioMoth (Open Acoustic Devices), was also used for recording. We used DeepSqueak software for most of the recording analysis and, in some cases, we also undertook further spectral analysis using RX8 (iZotope, USA). Results: We found that both systems can detect a range of ultrasonic noise sources, and here discuss the benefits and limitations of each approach. Conclusions: We conclude that measuring the acoustic environment of animal facilities, including ultrasonic frequencies that may adversely affect the animals housed, will contribute to minimising disruption to animal welfare and perturbations in scientific research.

Abstract 420: Proprotein Convertase Subtilisin/Kexin 6 Is Involved In Lipid Metabolism In Liver

Introduction: PCSK6 is a protease that activates cytokines and growth factors and strongly enriched in healthy human liver, however its function in this context has not been explored. We have previously shown that PCSK6 is induced in atherosclerotic plaques from patients with symptoms of stroke and important for regulating several cell types in this context. Here, we aimed to investigate the role of PCSK6 in lipid metabolism in liver, particularly in the context of atherosclerosis and non-alcoholic fatty liver disease (NAFLD). Methods: We used publically available datasets and several atherosclerosis biobanks to investigate the expression of PCSK6 in healthy and diseased human tissues. In addition, we used full Pcsk6 -/- mice as well as liver specific conditional Pcsk6 -/- knockout mice compared to littermate controls, to investigate the effects of PCSK6 ablation on lipid metabolism. Results: Genetic analyses of the PCSK6 locus identified a variant, rs7181043, that was significantly associated with PCSK6 mRNA expression in healthy human adipose tissue, liver and in atherosclerotic plaques. The same variant was associated specifically with plaque fat content and atherosclerotic patient’s plasma LDL levels. In addition, PCSK6 mRNA expression in plaques was positively correlated with total plasma cholesterol and LDL levels in atherosclerotic patients as well as lipid metabolism associated pathways within the carotid plaque. Microarray comparison of the livers from Pcsk6 -/- mice and controls showed that VLDL particle assembly was one of the upregulated processes. I n vivo studies showed that Pcsk6 -/- mice have higher plasma cholesterol and LPL levels at baseline compared to controls, and lower levels of LDLR in their liver. These findings were further confirmed in liver specific conditional knockouts. Preliminary results show that liver specific knockout mice develop increased liver steatosis and fibrosis on a modified western diet. Conclusions: Our data suggests that PCSK6 is involved in cholesterol and metabolic control in liver. Breeding of liver specific Pcsk6 knockout mice on an ApoE -/- background is currently ongoing and will provide insight into the role of liver PCSK6 in atherosclerosis and NAFLD development.

Identification and characterisation of spontaneous mutations causing deafness from a targeted knockout programme

BackgroundMice carrying targeted mutations are important for investigating gene function and the role of genes in disease, but off-target mutagenic effects associated with the processes of generating targeted alleles, for instance using Crispr, and culturing embryonic stem cells, offer opportunities for spontaneous mutations to arise. Identifying spontaneous mutations relies on the detection of phenotypes segregating independently of targeted alleles, and having a broad estimate of the level of mutations generated by intensive breeding programmes is difficult given that many phenotypes are easy to miss if not specifically looked for. Here we present data from a large, targeted knockout programme in which mice were analysed through a phenotyping pipeline. Such spontaneous mutations segregating within mutant lines may confound phenotypic analyses, highlighting the importance of record-keeping and maintaining correct pedigrees.ResultsTwenty-five lines out of 1311 displayed different deafness phenotypes that did not segregate with the targeted allele. We observed a variety of phenotypes by Auditory Brainstem Response (ABR) and behavioural assessment and isolated eight lines showing early-onset severe progressive hearing loss, later-onset progressive hearing loss, low frequency hearing loss, or complete deafness, with vestibular dysfunction. The causative mutations identified include deletions, insertions, and point mutations, some of which involve new genes not previously associated with deafness while others are new alleles of genes known to underlie hearing loss. Two of the latter show a phenotype much reduced in severity compared to other mutant alleles of the same gene. We investigated the ES cells from which these lines were derived and determined that only one of the 8 mutations could have arisen in the ES cell, and in that case, only after targeting. Instead, most of the non-segregating mutations appear to have occurred during breeding of mutant mice. In one case, the mutation arose within the wildtype colony used for expanding mutant lines.ConclusionsOur data show that spontaneous mutations with observable effects on phenotype are a common side effect of intensive breeding programmes, including those underlying targeted mutation programmes. Such spontaneous mutations segregating within mutant lines may confound phenotypic analyses, highlighting the importance of record-keeping and maintaining correct pedigrees.