Project description:MicroRNAs (miRNAs) are evolutionary conserved, non-coding, gene regulatory RNA molecules found in both plants and animals and amongst almost every cell and tissue type. They are about 22 nucleotides long and are involved in silencing of mRNA through sequence specific binding to the 3’ untranslated region (UTR) of the mRNA subsequently causing translational repression and/or will promote the degradation of protein-coding mRNA. Specifically, the miRNA family, mir-146 a/b, has been previously found to be involved in the regulation of the innate immune response by functioning as a negative regulator to help fine-tune the immune response. Microglial cells are the macrophage of the brain participating as major players of the innate immune response. During prion disease, no immune response is mounted against PrPSc possibly due to its similarity to host PrPc and thus, the host immune response would be suppressed and tightly regulated. Therefore, an increased expression of mir-146 by microglial cells during prion disease may function as one of these negative regulators. Our objective of this experiment is to use DNA microarrays to investigate the gene regulation of mir-146 previously found upregulated in our studies of mouse brain tissue specifically in microglial cells during prion disease with the aim of having a better understanding of prion pathobiology and a potential target for therapeutic intervention. Mir-146 expression was confirmed via in situ analysis of brain tissue and was further investigated in a microglial murine cell line (EOC 13.31). To mimic mir-146 upregulation during prion disease, mir-146 a & b were overexpressed in EOCs using a lipid based reverse transfection system. Furthermore, endogeneous mir-146 was knocked down as another method to help confirm the mRNA targets affected by mir-146. Simultaneously, another experiment was performed for the investigation of its involvement in the innate immune response by stimulating the EOCs with differing concentrations of LPS from E.coli (055:B5). Total RNA was collected and prepared at several timepoints and the levels of expression of both mir-146 a & b was tested via qRT-PCR. The RNA collected from the EOCs from each condition were used as target material for dual-color, competitive hybridization to Agilent whole mouse genome 4x44K oligo arrays. All the significant targets found on the mircorarrays were compared against the various conditions to find a consensus of affected mir-146 mRNA targets.
Project description:MicroRNAs (miRNAs) are evolutionary conserved, non-coding, gene regulatory RNA molecules found in both plants and animals and amongst almost every cell and tissue type. They are about 22 nucleotides long and are involved in silencing of mRNA through sequence specific binding to the 3’ untranslated region (UTR) of the mRNA subsequently causing translational repression and/or will promote the degradation of protein-coding mRNA. Specifically, the miRNA family, mir-146 a/b, has been previously found to be involved in the regulation of the innate immune response by functioning as a negative regulator to help fine-tune the immune response. Microglial cells are the macrophage of the brain participating as major players of the innate immune response. During prion disease, no immune response is mounted against PrPSc possibly due to its similarity to host PrPc and thus, the host immune response would be suppressed and tightly regulated. Therefore, an increased expression of mir-146 by microglial cells during prion disease may function as one of these negative regulators. Our objective of this experiment is to use DNA microarrays to investigate the gene regulation of mir-146 previously found upregulated in our studies of mouse brain tissue specifically in microglial cells during prion disease with the aim of having a better understanding of prion pathobiology and a potential target for therapeutic intervention.
Project description:Prion diseases typically have long pre-clinical incubation periods during which time the infectious prion particle and infectivity steadily propagate in the brain. Abnormal neuritic sprouting and synaptic deficits are apparent during pre-clinical disease, however, gross neuronal loss is not detected until the onset of the clinical phase. The molecular events that accompany early neuronal damage and ultimately conclude with neuronal death remain obscure. In this study, we used laser capture microdissection to isolate hippocampal CA1 neurons and then determined their pre-clinical transcriptional response during infection. We found that gene expression within these neurons is dynamic and characterized by distinct phases of activity. A major cluster of genes is altered during pre-clinical disease after which expression either returns to basal levels, or alternatively undergoes a direct reversal during clinical disease. Strikingly, we show that this cluster contains a signature highly reminiscent of synaptic N-methyl-D-aspartic acid (NMDA) receptor signaling and the activation of neuroprotective pathways. Additionally, genes involved in neuronal projection and dendrite development were also altered throughout the disease, culminating in a general decline of gene expression for synaptic proteins. Similarly, deregulated miRNAs such as miR-132-3p, miR-124a-3p, miR-16-5p, miR-26a-5p, miR-29a-3p and miR-140-5p follow concomitant patterns of expression. This is the first in depth genomic study describing the pre-clinical response of hippocampal neurons to early prion replication. Our findings suggest that prion replication results in the persistent stimulation of a programmed response, at least in part mediated by synaptic NMDA receptor activity that initially promotes cell survival and neurite remodelling. However, this response is terminated prior to the onset of clinical symptoms in the infected hippocampus, seemingly pointing to a critical juncture in the disease. Manipulation of these early neuroprotective pathways may redress the balance between degeneration and survival, providing a potential inroad for treatment. The CA1 hippocampal region was dissected out using LCM and RNA was extracted from these samples. In total, 6 different time points were screened for both RNA and miRNA expression levels in prion infected and control animals. Gene expression profiles from 6 time points (n M-bM-^IM-% 2) were determined using whole mouse 4x44K arrays. We successfully validated a subset of candidate genes that were deregulated during early prion disease. We performed a similar assessment of temporal miRNAs expression levels throughout the infection using the TLDA platform which was further validated by individual real-time PCR assays. In parallel, immunoctyochemistry was used to characterize the cellular presence of astrocytes, microglial and neurons in the CA1 region throughout the disease which correlated well with both mRNA and miRNA expression profiles. Staining for the PrPRes and neuronal toxicity levels was also performed to determine the spatial and temporal PrPRes deposition and assess the level of neuronal death that occurs in the hippocampus, respectively. Using bioinformatic methods, potential pathways that were implicated by our data to be deregulated during early prion disease were presented while potential miRNA regulation of some of these candidate genes implicated in these pathways was also included.
Project description:Introgressed variants from other species can be an important source of genetic variation because they may arise rapidly, can include multiple mutations on a single haplotype, and have often been pretested by selection in the species of origin. Although introgressed alleles are generally deleterious, several studies have reported introgression as the source of adaptive alleles-including the rodenticide-resistant variant of Vkorc1 that introgressed from Mus spretus into European populations of Mus musculus domesticus. Here, we conducted bidirectional genome scans to characterize introgressed regions into one wild population of M. spretus from Spain and three wild populations of M. m. domesticus from France, Germany, and Iran. Despite the fact that these species show considerable intrinsic postzygotic reproductive isolation, introgression was observed in all individuals, including in the M. musculus reference genome (GRCm38). Mus spretus individuals had a greater proportion of introgression compared with M. m. domesticus, and within M. m. domesticus, the proportion of introgression decreased with geographic distance from the area of sympatry. Introgression was observed on all autosomes for both species, but not on the X-chromosome in M. m. domesticus, consistent with known X-linked hybrid sterility and inviability genes that have been mapped to the M. spretus X-chromosome. Tract lengths were generally short with a few outliers of up to 2.7 Mb. Interestingly, the longest introgressed tracts were in olfactory receptor regions, and introgressed tracts were significantly enriched for olfactory receptor genes in both species, suggesting that introgression may be a source of functional novelty even between species with high barriers to gene flow.
Project description:Translational research is commonly performed in the C57B6/J mouse strain, chosen for its genetic homogeneity and phenotypic uniformity. Here, we evaluate the suitability of the white-footed deer mouse (Peromyscus leucopus) as a model organism for aging research, offering a comparative analysis against C57B6/J and diversity outbred (DO) Mus musculus strains. Our study includes comparisons of body composition, skeletal muscle function, and cardiovascular parameters, shedding light on potential applications and limitations of P. leucopus in aging studies. Notably, P. leucopus exhibits distinct body composition characteristics, emphasizing reduced muscle force exertion and a unique metabolism, particularly in fat mass. Cardiovascular assessments showed changes in arterial stiffness, challenging conventional assumptions and highlighting the need for a nuanced interpretation of aging-related phenotypes. Our study also highlights inherent challenges associated with maintaining and phenotyping P. leucopus cohorts. Behavioral considerations, including anxiety-induced responses during handling and phenotyping assessment, pose obstacles in acquiring meaningful data. Moreover, the unique anatomy of P. leucopus necessitates careful adaptation of protocols designed for Mus musculus. While showcasing potential benefits, further extensive analyses across broader age ranges and larger cohorts are necessary to establish the reliability of P. leucopus as a robust and translatable model for aging studies.
Project description:BackgroundCopy number variation is an important dimension of genetic diversity and has implications in development and disease. As an important model organism, the mouse is a prime candidate for copy number variant (CNV) characterization, but this has yet to be completed for a large sample size. Here we report CNV analysis of publicly available, high-density microarray data files for 351 mouse tail samples, including 290 mice that had not been characterized for CNVs previously.ResultsWe found 9634 putative autosomal CNVs across the samples affecting 6.87% of the mouse reference genome. We find significant differences in the degree of CNV uniqueness (single sample occurrence) and the nature of CNV-gene overlap between wild-caught mice and classical laboratory strains. CNV-gene overlap was associated with lipid metabolism, pheromone response and olfaction compared to immunity, carbohydrate metabolism and amino-acid metabolism for wild-caught mice and classical laboratory strains, respectively. Using two subspecies of wild-caught Mus musculus, we identified putative CNVs unique to those subspecies and show this diversity is better captured by wild-derived laboratory strains than by the classical laboratory strains. A total of 9 genic copy number variable regions (CNVRs) were selected for experimental confirmation by droplet digital PCR (ddPCR).ConclusionThe analysis we present is a comprehensive, genome-wide analysis of CNVs in Mus musculus, which increases the number of known variants in the species and will accelerate the identification of novel variants in future studies.
Project description:BackgroundLong terminal repeat (LTR) retrotransposons make up a large fraction of the typical mammalian genome. They comprise about 8% of the human genome and approximately 10% of the mouse genome. On account of their abundance, LTR retrotransposons are believed to hold major significance for genome structure and function. Recent advances in genome sequencing of a variety of model organisms has provided an unprecedented opportunity to evaluate better the diversity of LTR retrotransposons resident in eukaryotic genomes.ResultsUsing a new data-mining program, LTR_STRUC, in conjunction with conventional techniques, we have mined the GenBank mouse (Mus musculus) database and the more complete Ensembl mouse dataset for LTR retrotransposons. We report here that the M. musculus genome contains at least 21 separate families of LTR retrotransposons; 13 of these families are described here for the first time.ConclusionsAll families of mouse LTR retrotransposons are members of the gypsy-like superfamily of retroviral-like elements. Several different families of unrelated non-autonomous elements were identified, suggesting that the evolution of non-autonomy may be a common event. High sequence similarity between several LTR retrotransposons identified in this study and those found in distantly-related species suggests that horizontal transfer has been a significant factor in the evolution of mouse LTR retrotransposons.
Project description:House mice (Mus musculus) emit ultrasonic vocalizations (USVs), which are surprisingly complex and have features of bird song, but their functions are not well understood. Previous studies have reported mixed evidence on whether there are sex differences in USV emission, though vocalization rate or other features may depend upon whether potential receivers are of the same or opposite sex. We recorded the USVs of wild-derived adult house mice (F1 of wild-caught Mus musculus musculus), and we compared the vocalizations of males and females in response to a stimulus mouse of the same- or opposite-sex. To detect and quantify vocalizations, we used an algorithm that automatically detects USVs (Automatic Mouse Ultrasound Detector or A-MUD). We found high individual variation in USV emission rates (4 to 2083 elements/10 min trial) and a skewed distribution, with most mice (60%) emitting few (≤50) elements. We found no differences in the rates of calling between the sexes overall, but mice of both sexes emitted vocalizations at a higher rate and higher frequencies during opposite- compared to same-sex interactions. We also observed a trend toward higher amplitudes by males when presented with a male compared to a female stimulus. Our results suggest that mice modulate the rate and frequency of vocalizations depending upon the sex of potential receivers.
Project description:The aim of this study was to assess whether chronic treatment with RPV can modulate the progression of chronic liver disease, especially of non-alcoholic fatty liver disease (NAFLD), through a nutritional model in wild-type mice Mice were daily treated with RPV (p.o.) and fed with normal or high fat diet during 3 months to induce fatty liver disease