Transcription profiling of rat forebrain after exposure to novel environment
ABSTRACT: Expression profiling in whole rat forebrain in response to exposure of animals to a novel environment; Experiments were conducted to identify genes upregulated in response to novel sensory experiences. Experiment Overall Design: Rats were either housed in a standard laboratory cage (control) or exposed to a novel environment containing objects of different shapes and colors (novel environment) for three hours.
Project description:Expression profiling in whole rat forebrain in response to exposure of animals to a novel environment Experiments were conducted to identify genes upregulated in response to novel sensory experiences. Keywords: Gene expression profiling Overall design: Rats were either housed in a standard laboratory cage (control) or exposed to a novel environment containing objects of different shapes and colors (novel environment) for three hours.
Project description:This study dermined the temporal gene expression profile of myeloid subpopulations recruited to the peritoneal cavity to encapsulate implanted foreign material. Sterile foreign objects were inserted into the peritoneal cavities of MacGreen mice (in which the Csf1r promoter directs myeloid-specific EGFP expression). At various time-points post-surgery (days 2, 4, 7, 14), mice were euthanased, and peritoneal exudate cells removed by lavage. Peritoneal exudate cells from MacGreen mice that had not received implants were used as controls (day 0; 'unstimulated'). Free-floating objects encapsulated with tissue were removed from the peritoneal cavities of different mice at days 7, 14, 21, 28 days, and single cell suspensions obtained by collagenase digestion. Single-cell suspensions from peritoneal exudate or tissue capsules were separated on the basis of size/granularity and EGFP fluorescence using FacsVantage SE Diva. Total RNA was extracted from FACS-sorted EGFP-hi cells.
Project description:Gene-environment interactions mediated at the epigenetic level may provide an initial step in delivering an appropriate response to environmental changes. 5-hydroxymethylcytosine (5hmC), a DNA base derived from 5-methylcytosine (5mC), accounts for ~40% of modified cytosine in brain and has been implicated in DNA methylation-related plasticity. To identify the role of 5hmC in gene-environment interactions, we exposed both young (6-week-old) and aged (18-month-old) mice to both an enriched environment and a standard environment. Exposure to EE significantly improves learning and memory in aged mice and reduces 5hmC abundance in mouse hippocampus. Furthermore, we mapped the genome-wide distribution of 5hmC and found that the alteration of 5hmC modification occurred mainly at gene bodies. In particular, genes involved in axon guidance are enriched among the genes with altered 5hmC modification. These results together suggest that environmental enrichment could modulate the dynamics of 5hmC in hippocampus, which could potentially contribute to improved learning and memory in aged animals. To identify the role of 5hmC in gene-environment interactions, we exposed both young (6-week-old) and aged (18-month-old) C57B/6 mice to both an enriched environment (EE) and a standard environment. Exposure of mice to EE was achieved by keeping a group of mice in the EE chamber, which is larger than the standard cage, includes novel objects, such as toys of varied color and texture, tunnels, an exercise wheel for voluntary exercise, and extra bedding material, along with free access to food and water. Daily exposure to EE was kept at 5 hours during the daylight cycle for 4 consecutive weeks. Control animals were kept in their small cages that are used as standard housing cages (CE) containing bedding, food and access to water. YC, young mice exposing to control environment, YE, young mice exposing to enriched environment, AC, aged mice exposing to control environment, AE, aged mice exposed to enriched environment. Please note that 5hmC-containing DNA enrichment method was inspired by a unique character of beta-glucotransferase that can specifically add glucose to 5hmC modification. With a modified glucose conjugated with biotin, we are able to purify the 5hmC-containing DNA by biotin-streptavidin-based immunoprecipitation.
Project description:We used microarrays to assess gene expression differences in the hippocampus between FoxO6 mutant and wild-type siblings before (basal) or after novel object learning. The cohort of basal mice were housed individually for at least 3 days prior to tissue harvesting. The mice used to collect hippocampal samples after the object learning task were handled daily in the procedure room, prior to the object learning task. 24 hours before the object learning task, each mouse was individually habituated to the empty novel object arena for 10 min. On the days of the novel object learning task and the empty arena habituation, the mice were transferred to the procedure room and acclimatized for 60 min. For the novel object learning task, the mice were allowed to explore two identical and novel objects for 10 min in a 70 x 70 x 70 cm black plastic arena with a white PVC vinyl material on the base. After the object exploration, mice were transferred to an empty cage, and were euthanized after 60 min. The brains were dissected and incubated in ice-cold RNAlater (Ambion) for 24 hours at 4ºC. The brains were then rapidly frozen in O.C.T. Tissue-Tek (Sakura) at -80ºC. Coronal brain sections (300 µm) were made using a microtome (Microm), the hippocampus was finely dissected and collected into ‘RNA lysis buffer’ (Ambion), and homogenized for 30 sec using a rotar-stat homogenizer. Total RNA was extracted using the RNAqueous column (Ambion) following the manufacturer’s protocol.
Project description:It is possible to identify the key genes and pathways involved in specific physiological processes using transcriptome analyses. However, these powerful new deep sequencing-based methods have rarely been applied to studies of memory function. We used the bow-tie maze to train rats by exposing them to highly familiar objects or to novel objects. Total RNA sequencing was then used to compare the transcriptome of the perirhinal cortices of naïve control rats and rats exposed to novel and familiar stimuli. Differentially expressed genes were identified between group Novel and group Familiar rats and these included genes coding for transcription factors and extracellular matrix-related proteins. Moreover, differences in alternative splicing were also detected between the two groups. To conclude, this study shows that RNA sequencing can be used as a tool to identify differences in gene expression in behaving animals undergoing the same task but encountering different exposures. Overall design: RNA profiles of perirhinal cortex from rats exposed to novel objects (n=5) or familiar objects (n=5) in a recognition memory task were investigated using the Ion Proton System. Controls were naïve rats that had not undergone any behavioural testing (n=4).
Project description:6-year-old ramets of U. minor (Atinian elm clone) were inoculated with O. novo-ulmi in order to evaluate molecular responses activated during plant colonization. To elucidate the different genes involved in the U. minor immune system and the molecular changes suffered after inoculation, oligomicroarrays were constructed using the data from the transcriptome available in the Dryad database (Perdiguero et al., 2015; http://dx.doi.org/10.5061/dryad.ps837), and hybridized with cDNA obtained from the ramets over a time course following inoculation. Three biological replicates for control and inoculated plants for each sampling point (1, 3, 7, 14 and 21 days post-inoculation) were hybridised using two colors (inoculated vs control).
Project description:Background: In a recent intervention study, the daily supplementation with 200mg monomeric and oligomeric flavanols (MOF) from grape seeds for 8 weeks revealed a vascular health benefit in male smokers. The objective of the present study was to determine the impact of MOF consumption on the gene expression profile of leukocytes and to assess changes in DNA methylation. Methodology/Principal Findings: Gene expression profiles were determined using whole genome microarrays (Agilent) and DNA methylation was assessed using HumanMethylation450 BeadChips (Illumina). MOF significantly modulated the expression of 869 genes. The majority of the affected genes are involved in chemotaxis, cell adhesion, cell infiltration or cytoskeleton organisation, suggesting lower immune cell adhesion to endothelial cells. This was corroborated by in vitro experiments showing that MOF exposure of monocytes attenuates their adhesion to TNFα-stimulated endothelial cells. Nuclear factor-kappa B reporter gene assays confirmed that MOF decrease the activity of NF-κB. Strong inter-individual variability in the leukocytes DNA methylation was observed. As a consequence, on group level, changes due to MOF supplementation could not be found. However, in individuals, significant changes in DNA methylation of genes involved in leukocyte rolling, adhesion and cytoskeleton remodelling were seen. At the group level, the variation in DNA methylation related to life style and clinical parameters appeared to overrule the magnitude of effects of supplementation with MOF. Conclusion: Our study revealed that regular consumption of MOF modulates the expression of genes in immune cells which, however, cannot be correlated to alterations in the DNA methylome. Remarkably, at the individual level, genes related to leukocyte adhesion pathways present complex variation in DNA methylation. As such, the individual transcriptional and epigenetic modulation of genes involved in early manifestation of cardiovascular diseases constitutes important subcellular mechanisms by which MOF promote vascular health in humans. Two-colors experiment, 7 volunteers : blood samples of 7 men, before and after an intervention of 8 weeks with daily intake of MOF (14 samples).
Project description:We examined the transcriptional responses of intracellular M. tuberculosis exposed to different front-line drugs during macrophage infection. Our data reveal that Mtb experiences amplification of multiple host-derived pressures following drug exposure. The stresses from the host environment have a dominant impact on Mtb gene expression to the extent that the transcriptional profiles cluster according to environment, ie. broth vs host cell, rather than drug. Overall design: Transcriptional responses of M. tuberculosis to front-line drugs were studied under two environmental conditions: J774 macrophages vs. 7H9 broth, and 4 drugs were examined: INH, RIF, EMB, and PZA.
Project description:This microarray study aimed at evaluating the impact of mosquito chemical environment on the selection of insecticide resistance mechanisms. Here the mosquito Aedes aegypti was used as a model to perform a laboratory experiment combining mosquito larvae exposure to a sub-lethal dose of xenobiotic and their selection with the insecticide permethrin. After ten generations, bioassays and a transcriptome profiling with the 15K microarray Aedes detox chip plus microarray were performed comparatively on all strains.