Mouse Microarrays Research Articles

The screening of a microRNA expression during development of human macrophages and mouse dendritic cells

ABSTRACTThere is increasing evidence showing specific roles of microRNA in cell differentiation and cancer progression. Here we examine miRNA profiles during maturation of monocytes and bone marrow-derived dendritic cells (BMDCs) in human and mouse, respectively. We have identified significant changes of various miRNA expression during monocyte and BMDC monocyte development via miRNA microarrays, confirmed by quantitative PCR. Increases in miR155 expression positively correlated with increasing maturity of monocyte and BMDC in both mouse and human microarrays, indicating its importance in development. We describe a requirement of miR155 for MHCII expression during GM-CSF-induced development and LPS-induced maturation of DCs, suggesting reduced immune function of DC when miR155 is absent. Our study suggests that miRNAs might have an important role in differentiation of myeloid cell such as dendritic cells and macrophages.

Cross-species hybridization of foot-and-mouth disease virus-infected BHK-21 cells using human and mouse oligonucleotide microarrays.

Foot-and-mouth disease virus (FMDV) has a dual capacity to induce either acute or persistent infection in host animals. Establishment of an in vitro cell model of FMDV persistent infection facilitates the study of the mechanism underlying this type of infection. In this study, we analyzed gene expression profiles of both acute and persistent infections using cross-species microarrays. Our data suggest that human microarrays are more efficient than mouse microarrays in hybridization with cDNA from BHK-21 cells although the mouse is closer to the Syrian hamster in taxonomy. A set of differentially expressed genes (DEGs) that may be involved in the determination of acute or persistent infection was identified by using human or mouse microarrays. Seven common DEGs were found in both human and mouse arrays and showed similar fold changes. Among the DEGs, 33 genes were selected for further validation by using qRT-PCR and presented consistent results. The analysis of Gene Ontology Biological Processes indicated that various biosynthetic and metabolic processes were negatively regulated in the group of acute infection whereas multicellular organismal development processes were positively regulated in the group of persistent infection. Our study demonstrates the plausibility and utility of using cross-species microarrays to study FMDV-infected mammalian cells. The combined use of two types of microarrays can be more informative in exploring the mechanisms underlying the infections of FMDV.

Mechanisms of prostate carcinogenesis and its prevention by a γ-tocopherol-rich mixture of tocopherols in TRAMP mice

Tocopherols belong to a subgroup of the vitamin E family. Dietary feeding of a γ-tocopherol-rich mixture of tocopherols(γ-Tm T) inhibits prostate tumorigenesis in TRAMP mice. In this study, we aimed to investigate mechanisms of prostate carcinogenesis in TRAMP mice by identifying differentially expressed pathways and effects of γ-Tm T on these pathways. Eight-week-old TRAMP and age-matched C57BL/6 mice were administered either 600 mg/kg of γ-Tm T or a control vehicle via oral gavage. Twelve hours after dosing, prostate tissues were collected for RNA extraction. Whole genome mouse microarrays were used to examine gene expression profiles. The expression of the selected genes were validated using quantitative PCR. Thousands of genes and various pathways were altered in the prostates of TRAMP mice. Compared to C57BL/6 mice, TRAMP mice exhibited enhanced proliferation, suppressed expression of antioxidant and phase II detoxification enzymes, and metabolic reprogramming in the prostate. γ-Tm T differentially regulated the gene expression profiles of TRAMP and C57BL/6 mice, with only a small percentage of genes overlapping. γ-Tm T inhibited genes involved in proliferation and glucose metabolism and induced several antioxidant/phase II detoxification genes in TRAMP mice. γ-Tm T modulates multiple aberrant pathways in the prostate of TRAMP mice, which might represent important mechanisms for prostate cancer prevention.

Genomic profiling of a Hepatocyte growth factor-dependent signature for MET-targeted therapy in glioblastoma.

BackgroundConstitutive MET signaling promotes invasiveness in most primary and recurrent GBM. However, deployment of available MET-targeting agents is confounded by lack of effective biomarkers for selecting suitable patients for treatment. Because endogenous HGF overexpression often causes autocrine MET activation, and also indicates sensitivity to MET inhibitors, we investigated whether it drives the expression of distinct genes which could serve as a signature indicating vulnerability to MET-targeted therapy in GBM.MethodsInterrogation of genomic data from TCGA GBM (Student’s t test, GBM patients with high and low HGF expression, p ≤ 0.00001) referenced against patient-derived xenograft (PDX) models (Student’s t test, sensitive vs. insensitive models, p ≤ 0.005) was used to identify the HGF-dependent signature. Genomic analysis of GBM xenograft models using both human and mouse gene expression microarrays (Student’s t test, treated vs. vehicle tumors, p ≤ 0.01) were performed to elucidate the tumor and microenvironment cross talk. A PDX model with EGFRamp was tested for MET activation as a mechanism of erlotinib resistance.ResultsWe identified a group of 20 genes highly associated with HGF overexpression in GBM and were up- or down-regulated only in tumors sensitive to MET inhibitor. The MET inhibitors regulate tumor (human) and host (mouse) cells within the tumor via distinct molecular processes, but overall impede tumor growth by inhibiting cell cycle progression. EGFRamp tumors undergo erlotinib resistance responded to a combination of MET and EGFR inhibitors.ConclusionsCombining TCGA primary tumor datasets (human) and xenograft tumor model datasets (human tumor grown in mice) using therapeutic efficacy as an endpoint may serve as a useful approach to discover and develop molecular signatures as therapeutic biomarkers for targeted therapy. The HGF dependent signature may serve as a candidate predictive signature for patient enrollment in clinical trials using MET inhibitors. Human and mouse microarrays maybe used to dissect the tumor-host interactions. Targeting MET in EGFRamp GBM may delay the acquired resistance developed during treatment with erlotinib.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0667-x) contains supplementary material, which is available to authorized users.

Widespread cerebellar transcriptome changes in Ts65Dn Down syndrome mouse model after lifelong running

Our previous study showed an improvement in locomotor deficits after voluntary lifelong running in Ts65Dn mice, an animal model for Down syndrome (DS). In the present study, we employed mouse microarrays printed with 55,681 probes in an attempt to identify molecular changes in the cerebellar transcriptome that might contribute to the observed behavioral benefits of voluntary long-term running in Ts65Dn mice. Euploid mice were processed in parallel for comparative purposes in some analyses. We found that running significantly changed the expression of 4,315 genes in the cerebellum of Ts65Dn mice, over five times more than in euploid animals, up-regulating 1,991 and down-regulating 2,324 genes. Functional analysis of these genes revealed a significant enrichment of 92 terms in the biological process category, including regulation of biosynthesis and metabolism, protein modification, phosphate metabolism, synaptic transmission, development, regulation of cell death/apoptosis, protein transport, development, neurogenesis and neuron differentiation. The KEGG pathway database identified 18 pathways that are up-regulated and two that are down-regulated by running that were associated with learning, memory, cell signaling, proteolysis, regeneration, cell cycle, proliferation, growth, migration, and survival. Of six mRNA protein products we tested by immunoblotting, four showed significant running-associated changes in their levels, the most prominent in glutaminergic receptor metabotropic 1, and two showed changes that were close to significant. Thus, unexpectedly, our data point to the high molecular plasticity of Ts65Dn mouse cerebellum, which translated into humans with DS, suggests that the motor deficits of individuals with DS could markedly benefit from prolonged exercise

Crtc1 activates a transcriptional program deregulated at early Alzheimer's disease-related stages.

Cognitive decline is associated with gene expression changes in the brain, but the transcriptional mechanisms underlying memory impairments in cognitive disorders, such as Alzheimer's disease (AD), are largely unknown. Here, we aimed to elucidate relevant mechanisms responsible for transcriptional changes underlying early memory loss in AD by examining pathological, behavioral, and transcriptomic changes in control and mutant β-amyloid precursor protein (APPSw,Ind) transgenic mice during aging. Genome-wide transcriptome analysis using mouse microarrays revealed deregulation of a gene network related with neurotransmission, synaptic plasticity, and learning/memory in the hippocampus of APPSw,Ind mice after spatial memory training. Specifically, APPSw,Ind mice show changes on a cAMP-responsive element binding protein (CREB)-regulated transcriptional program dependent on the CREB-regulated transcription coactivator-1 (Crtc1). Interestingly, synaptic activity and spatial memory induces Crtc1 dephosphorylation (Ser151), nuclear translocation, and Crtc1-dependent transcription in the hippocampus, and these events are impaired in APPSw,Ind mice at early pathological and cognitive decline stages. CRTC1-dependent genes and CRTC1 levels are reduced in human hippocampus at intermediate Braak III/IV pathological stages. Importantly, adeno-associated viral-mediated Crtc1 overexpression in the hippocampus efficiently reverses Aβ-induced spatial learning and memory deficits by restoring a specific subset of Crtc1 target genes. Our results reveal a critical role of Crtc1-dependent transcription on spatial memory formation and provide the first evidence that targeting brain transcriptome reverses memory loss in AD.

Abstract 17984: Protective Role of Dicer in Macrophages During Atherogenesis

Atherosclerotic lesion development is characterized by the subendothelial accumulation of lipid-laden macrophages. It is known that microRNAs (miRs) are generated by the RNase Dicer and play important roles in macrophage function by negatively regulating post-transcriptional gene expression. Previously, we found that macrophage-derived miR-342-5p enhances atherosclerosis by fostering inflammatory macrophage activation. However, the impact of Dicer-dependent miR expression in macrophages during atherosclerosis is unclear. To study the role of Dicer in macrophages in atherosclerosis, we fed Apoe-/- mice with a myeloid cell-specific deletion of Dicer (LysMCre-Dicerflox) and littermate controls (LysMCre-DicerWT) a high cholesterol diet (HCD) for 3 months. Dicer deletion in myeloid cells was confirmed in bone marrow-derived macrophages isolated from LysMCre-Dicerflox mice by real-time PCR. The lipid deposition in the aortas was increased by 50% in LysMCre-Dicerflox (3.3±0.3%) compared to LysMCre-Dicerwt mice (2.1±0.3%) (p = 0.01, n = 13). The differential blood counts and the serum cholesterol and triglyceride levels were not altered between two groups. We found that 79 miRs, including those highly expressed in macrophages (e.g., miR-146a and miR-223), were down-regulated in LysMCre-Dicerflox aortas compared to controls after 3 months of a HCD (p < 0.05, n = 3) using a miR real-time PCR array. In aortas from LysMCre-Dicerflox mice, 471 genes were up-regulated as determined by whole-genome gene expression profiling using Agilent Mouse Microarrays (p < 0.05, n = 3-4). Genes involved in fatty acid and lipid metabolism were enriched among the up-regulated genes as analyzed by GeneGo MetaCore. Up-regulation of Plg, Ptges2, Eci1, Impa2, Acox2, Thrsp, and the lipid droplet-related genes plin-1, -2, -5 and Cidea was confirmed by real-time PCR. Whereas Plg was predicted to be a target of miR-223 by Targetscan, plin-1, -2, and Ptges2 were identified as potential targets of miR-146a using miRanda. These data indicate a protective role of Dicer during atherogenesis by inhibiting lipid metabolism in lesional macrophages through miRs. Thus, regulation of the lipid metabolism in macrophages might be a critical step in the development of atherosclerosis.

Thyroid hormone-regulated gene expression in juvenile mouse liver: identification of thyroid response elements using microarray profiling and in silico analyses

BackgroundDisruption of thyroid hormone signalling can alter growth, development and energy metabolism. Thyroid hormones exert their effects through interactions with thyroid receptors that directly bind thyroid response elements and can alter transcriptional activity of target genes. The effects of short-term thyroid hormone perturbation on hepatic mRNA transcription in juvenile mice were evaluated, with the goal of identifying genes containing active thyroid response elements. Thyroid hormone disruption was induced from postnatal day 12 to 15 by adding goitrogens to dams' drinking water (hypothyroid). A subgroup of thyroid hormone-disrupted pups received intraperitoneal injections of replacement thyroid hormones four hours prior to sacrifice (replacement). An additional group received only thyroid hormones four hours prior to sacrifice (hyperthyroid). Hepatic mRNA was extracted and hybridized to Agilent mouse microarrays.ResultsTranscriptional profiling enabled the identification of 28 genes that appeared to be under direct thyroid hormone-regulation. The regulatory regions of the genome adjacent to these genes were examined for half-site sequences that resemble known thyroid response elements. A bioinformatics search identified 33 thyroid response elements in the promoter regions of 13 different genes thought to be directly regulated by thyroid hormones. Thyroid response elements found in the promoter regions of Tor1a, 2310003H01Rik, Hect3d and Slc25a45 were further validated by confirming that the thyroid receptor is associated with these sequences in vivo and that it can bind directly to these sequences in vitro. Three different arrangements of thyroid response elements were identified. Some of these thyroid response elements were located far up-stream (> 7 kb) of the transcription start site of the regulated gene.ConclusionsTranscriptional profiling of thyroid hormone disrupted animals coupled with a novel bioinformatics search revealed new thyroid response elements associated with genes previously unknown to be responsive to thyroid hormone. The work provides insight into thyroid response element sequence motif characteristics.

Identification of pig reproductive QTL genes based on gene set enrichment analysis of mouse microarray dataset

Nowadays, abundantly different transcripts related to reproductive traits using mouse model have been identified by microarray analysis with the robustness, which were needed of re-evaluation with quantitive real time PCR (qPCR). Meanwhile, pig QTL databases were established but with large spans in the confidence interval for QTL location. Therefore, a genome-wide comparative and functional analysis of the association between mouse microarrays analysis results and reported pig QTLs is needed to better elucidate the genetic and physiological background of pig enhanced reproductive performance in genomics. Here, we employed a microarray dataset of a high fertility mouse line and applied gene set enrichment analysis method to identify significant mouse genes in a pathway level. Next we used a comparative genomic approach to find the homologous pig genes and locate them to the interval of the reported QTL for pigs’ traits based on AnimalQTLdb. Finally, we identified some pathways participating in pig high fertility, and some pig genes were further identified within the reported QTL location related to reproductive performance. Combining microarray analysis of mouse high fertility dataset by GSEA with pigQTLdb will substantially help us to identify candidate genes in reported QTL regions related to pig reproduction that are eventually responsible for increased fertility performance in pig and are also helpful for understanding the genetic information of pig reproduction in genomics. Furthermore, we can also provide some novel pig genes in identified pathways with relationship of reproduction. Key words: Reproductive, microarray, pig, pathway, endocrine.

Abstract 4867: Whole transcript expression microarray profiling of normal colon and adenocarcinoma total RNA

Abstract Whole transcript expression profiling by microarray analysis is an important tool for understanding biological mechanisms and development, classifying tissue and tumor types, and identifying indicators for diagnosis and prognosis. This analysis measures the alternative splicing of exons from a given RNA transcript leading to the translation of a variety of proteins from a single RNA transcript. To address the need for whole transcriptome expression profiling from low quantities of total RNA, we have developed an exon expression workflow which includes custom and catalog human, mouse, and rat exon microarrays, a whole transcript labeling method, and analysis software. This fast and simple microarray-based method employs a modified linear amplification procedure to generate Cy-labeled cRNA representing the whole transcript. The labeling method, available in the Low Input Quick Amp Labeling WT Kit (LIQA WT), employs a mixture of oligo dT- and random nucleotide-based T7 promoter primers (WT Primer Mix) resulting in high cRNA yields and specific activities from low RNA inputs. The probes on the human, mouse, and rat SurePrint G3 exon microarrays were designed from the high quality content of the public databases, primarily RefSeq and Ensembl. Exon array results are analyzed for gene and exon-level expression using GeneSpring GX 11.5 software. The exon gene expression workflow allowed whole transcript gene expression profile comparisons to be made in fewer than two days. Comparisons of probe signals from technical replicate samples demonstrated high reproducibility with a wide dynamic range and comparable signals across a broad range of input amounts. High correlations were demonstrated in platform comparisons to both qRT-PCR and RNA-Seq. This new workflow was used to detect alternative splicing of exons between cancerous and normal cells resulting in gene and exon expression profiles consistent with the current literature. These results demonstrate the value of the new exon expression workflow in identifying known exons, alternative start and stop sites, mutually exclusive exons, and cassette exons in cancer research. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4867. doi:10.1158/1538-7445.AM2011-4867

Chemostat‐based transcriptional analysis of growth rate change and BCL‐2 over‐expression in NS0 cells

We investigated the transcriptional response of NS0 cells undergoing controlled nutrient growth change in continuous chemostat culture using mouse microarrays. A 50% reduction in growth rate resulted in detectable alterations in the expression of 29 genes in NS0 cells. Notably, expression of genes in three major biological processes, namely transcriptional, translational, and protein processing functions, were modified. To further elucidate the advantage of the chemostat environment for establishment of "omic" data sets, an expression profile of the over-expressed gene bcl-2 in NS0 cells was probed. Functional analysis revealed that the underlying altered molecular mechanism was particularly associated with G1 cell cycle progression, protein synthesis, and apoptosis. Importantly, these findings agreed with the physical function of the cells. Despite an increase in survival rate, bcl-2 over-expression resulted in a decrease of specific productivity, glucose consumption, oxygen uptake rate and intracellular protein content, indicating a lower energy generating metabolism. Further, a prolongation of G1 cell cycle phase was evident on lowering the growth rate. Overall, the application of microarray analysis to chemostat-grown cultures offers an excellent combination for the interpretation of transcriptomic profiles to elucidate the molecular mechanisms during nutrient growth change and bcl-2 over-expression.

Transcription Profiles of Aortic Smooth Muscle Cells from Atherosclerosis-Prone and -Resistant Regions in Young Apolipoprotein E-Deficient Mice before Plaque Development

Background/Aims: Site-specific atherosclerosis is generally attributed to differential gene expression in endothelial cells. We investigated whether the transcriptome of smooth muscle cells is different between atherosclerosis-prone and atherosclerosis-resistant regions in apolipoprotein E-deficient (apoE–/–) mice before plaque development, and in C57Bl/6 mice. Methods: De-endothelialized aortas (both strains: 3 males, 3 females, age 4 months) were divided into atherosclerosis-prone (AA: ascending aorta, aortic arch and proximal 2 mm of thoracic aorta) and -resistant (CTA: central thoracic aorta, i.e. 6 mm distal from the proximal 2 mm) regions. The transcriptome of these two regions was compared using whole-genome mouse microarrays. Results: Microarray analysis revealed differential expression (>2-fold difference) of 70 and 244 genes in C57Bl/6 and apoE–/– mice. This was confirmed for 6 genes using the real-time quantitative polymerase chain reaction. Up- or downregulation in the AA was observed for 33 and 37 genes in C57Bl/6, and for 186 and 58 genes in apoE–/– mice, respectively. The 201 genes that showed exclusively differential expression in apoE–/– mice were related to atherosclerotic processes, such as cell adhesion, proliferation, differentiation, motility, cell death, lipid metabolism and immune responses. Conclusion: Our findings indicate that smooth muscle cells display an altered transcriptome at atherosclerosis-prone locations before actual lesion development.

Abstract 546: Characterization of tumor-stromal interactions in microenvironment of prostate cancer bone metastasis

Abstract Prostate cancer is the most common cancer in men around the world, with a high incidence of bone metastasis, which is the major determinant of mortality in prostate cancer. The survival and proliferation of prostate cancer cells metastatic to bone result from the cross-talk between carcinoma cells and the stromal cells. In contrast to skeletal metastases resulting from breast or lung cancer that are most often osteolytic, metastases from prostate carcinoma are nearly always osteoblastic, suggesting that interactions between prostate cancer cells and stroma in bone might be different from those of lung or breast cancer cells and bone stroma. Therefore, gene expression patterns that are unique to osteoblastic prostate cancer will help to identify genes involved in the bidirectional cross-talk between prostate cancer cells and bone cells in microenvironment niche. However, because gene expression signatures from human cancer tissues are a mixture of those from carcinoma and stromal cells, it has been impossible to delineate stromal cell specific gene expression signatures from carcinoma cell signatures. Thus, we have devised a novel experimental strategy: “competitive cross-species hybridization of microarray”, which can simultaneously extract gene expression patterns of carcinoma cells and stromal cells from a single xenograft sample. In the current study, we xenografted human prostate cancer cells (PC3, PCA118b, 10A) into mouse bone and prostate and used these tissues for microarray experiments. Total RNAs were purified from three different tissue groups: normal bones (mouse RNA only), tumor-bearing bones (mixture of mouse and human RNA), and tumor-bearing prostates (mixture of mouse and human RNA). Labeled cRNA from normal bones and tumor-bearing bones were hybridized to mouse microarray slide to measure gene expression of mouse bone cells. Likewise, labeled cRNA from tumor-bearing prostate and tumor-bearing bone were hybridized to human microarray slide to measure gene expression of human prostate cancer cells. Gene expression data from mouse microarrays identified gene expression patterns unique to bone cells that interact with prostate cancer cells in bone. When same analysis applied to human data, it also uncovered prostate cancer cell gene expression signatures associated with different microenvironment (prostate vs. bone). In order to uncover gene networks involved in cross-talk between prostate cancer cells and bone cells in metastatic bone microenvironment, gene network analysis using Ingenuity Pathway Analysis (IPA) was applied to combined gene lists from human and mouse data. In conclusion, by applying novel experimental strategy, “competitive cross-species hybridization of microarray”, we uncovered critical gene network involved in bidirectional cellular communication that drives the survival of prostate cancer cells in metastatic bone microenvironment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 546.

Cardiac gene expression and systemic cytokine profile are complementary in a murine model of post-ischemic heart failure

After myocardial infarction (MI), activation of the immune system and inflammatory mechanisms, among others, can lead to ventricular remodeling and heart failure (HF). The interaction between these systemic alterations and corresponding changes in the heart has not been extensively examined in the setting of chronic ischemia. The main purpose of this study was to investigate alterations in cardiac gene and systemic cytokine profile in mice with post-ischemic HF. Plasma was tested for IgM and IgG anti-heart reactive repertoire and inflammatory cytokines. Heart samples were assayed for gene expression by analyzing hybridization to AECOM 32k mouse microarrays. Ischemic HF significantly increased the levels of total serum IgM (by 5.2-fold) and total IgG (by 3.6-fold) associated with a relatively high content of anti-heart specificity. A comparable increase was observed in the levels of circulating pro-inflammatory cytokines such as IL-1beta (3.8X) and TNF-alpha (6.0X). IFN-gamma was also increased by 3.1-fold in the MI group. However, IL-4 and IL-10 were not significantly different between the MI and sham-operated groups. Chemokines such as MCP-1 and IL-8 were 1.4- and 13-fold increased, respectively, in the plasma of infarcted mice. We identified 2079 well annotated unigenes that were significantly regulated by post-ischemic HF. Complement activation and immune response were among the most up-regulated processes. Interestingly, 21 of the 101 quantified unigenes involved in the inflammatory response were significantly up-regulated and none were down-regulated. These data indicate that post-ischemic heart remodeling is accompanied by immune-mediated mechanisms that act both systemically and locally.

Shotgun Proteomics Analysis of Hibernating Arctic Ground Squirrels

Mammalian hibernation involves complex mechanisms of metabolic reprogramming and tissue protection. Previous gene expression studies of hibernation have mainly focused on changes at the mRNA level. Large scale proteomics studies on hibernation have lagged behind largely because of the lack of an adequate protein database specific for hibernating species. We constructed a ground squirrel protein database for protein identification and used a label-free shotgun proteomics approach to analyze protein expression throughout the torpor-arousal cycle during hibernation in arctic ground squirrels (Urocitellus parryii). We identified more than 3,000 unique proteins from livers of arctic ground squirrels. Among them, 517 proteins showed significant differential expression comparing animals sampled after at least 8 days of continuous torpor (late torpid), within 5 h of a spontaneous arousal episode (early aroused), and 1-2 months after hibernation had ended (non-hibernating). Consistent with changes at the mRNA level shown in a previous study on the same tissue samples, proteins involved in glycolysis and fatty acid synthesis were significantly underexpressed at the protein level in both late torpid and early aroused animals compared with non-hibernating animals, whereas proteins involved in fatty acid catabolism were significantly overexpressed. On the other hand, when we compared late torpid and early aroused animals, there were discrepancies between mRNA and protein levels for a large number of genes. Proteins involved in protein translation and degradation, mRNA processing, and oxidative phosphorylation were significantly overexpressed in early aroused animals compared with late torpid animals, whereas no significant changes at the mRNA levels between these stages had been observed. Our results suggest that there is substantial post-transcriptional regulation of proteins during torpor-arousal cycles of hibernation.

Dose-dependent transitions in Nrf2-mediated adaptive response and related stress responses to hypochlorous acid in mouse macrophages

Hypochlorous acid (HOCl) is potentially an important source of cellular oxidative stress. Human HOCl exposure can occur from chlorine gas inhalation or from endogenous sources of HOCl, such as respiratory burst by phagocytes. Transcription factor Nrf2 is a key regulator of cellular redox status and serves as a primary source of defense against oxidative stress. We recently demonstrated that HOCl activates Nrf2-mediated antioxidant response in cultured mouse macrophages in a biphasic manner. In an effort to determine whether Nrf2 pathways overlap with other stress pathways, gene expression profiling was performed in RAW 264.7 macrophages exposed to HOCl using whole genome mouse microarrays. Benchmark dose (BMD) analysis on gene expression data revealed that Nrf2-mediated antioxidant response and protein ubiquitination were the most sensitive biological pathways that were activated in response to low concentrations of HOCl (< 0.35 mM). Genes involved in chromatin architecture maintenance and DNA-dependent transcription were also sensitive to very low doses. Moderate concentrations of HOCl (0.35 to 1.4 mM) caused maximal activation of the Nrf2 pathway and innate immune response genes, such as IL-1β, IL-6, IL-10 and chemokines. At even higher concentrations of HOCl (2.8 to 3.5 mM) there was a loss of Nrf2-target gene expression with increased expression of numerous heat shock and histone cluster genes, AP-1-family genes, cFos and Fra1 and DNA damage-inducible Gadd45 genes. These findings confirm an Nrf2-centric mechanism of action of HOCl in mouse macrophages and provide evidence of interactions between Nrf2, inflammatory, and other stress pathways.

Quality assessment of cross‐species hybridization of CHO transcriptome on a mouse DNA oligo microarray

DNA microarray technology has been widely utilized for species with extensive genome sequence information available. Given the limited genomic information pertaining to Chinese hamster ovary (CHO) cell line, cross-species hybridization using mouse microarrays provides a viable alternative. In this study, the utility of mouse Affymetrix microarrays for transcriptome profiling in CHO cells was assessed by hybridizing identical sets of cRNA samples from CHO cells on both mouse and CHO Affymetrix microarrays. Expression level measured by probe sets for orthologous transcripts on the two microarrays was compared. Only a fraction of the orthologous probes which detected expression calls in same species hybridization were similarly called present in cross species hybridization. In further analysis at the 25-mer probe level, it was revealed that specific hybridization signals were detectable by the subset of mouse probes that have a high degree of homology to the corresponding CHO sequences. The feasibility of cross species hybridization for quantifying the extent of differential expression was assessed by comparing transcript levels of CHO cells cultivated with and without sodium butyrate. While same species hybridization gave consistent degree of differential expression calls in replicated runs, a much inferior ability in quantifying differential expression was seen with cross species hybridization. Our results demonstrate that through detailed analysis of homology at the probe pair level, a subset of probes on existing mouse Affymetrix oligo-array can be used successfully for transcriptome profiling of CHO cells.

Gene Expression Profile of 2058 Spermatogenesis-Related Genes in Mice

The aim of this study was to characterize the gene expression profiles of mouse testis at different stages by performing large-scale complementary DNA (cDNA) analysis using DNA microarrays. The transcription profile of mouse testis was determined by comparing testis samples of mice aged 4, 9, 18, 35, 54 d and 6 months using Affymetrix Genechip mouse microarrays (430 v.2). Of the six comparative pairs of two neighboring spots examined, lots of differently expressed genes existed on day 18 versus day 9 and on day 35 versus day 18, indicating these genes may be involved in unique events during those periods. In mining the Genechip data, the gene expression profiles of 2058 genes represented in a gradually increased manner in the developmental stages of mice testis, and its validity was confirmed by the data of 21 known spermatogenesis-related testis-specific genes in our chip and the results of 10 random sample from 2058 genes produced by the sub-quantitative RT-PCR. So, we called these 2058 genes spermatogenesis-related genes. By informatics analysis, the gene expression clustering, gene ontology, KEGG pathway and domain regions were predicted for describing the potential roles that may play during mouse spermatogenesis. This study provides a molecular basis for the identity of spermatogenesis-related gene in mouse testis and leads to the elucidation of the molecular events underlying mammalian male reproduction.

Neonatal dopamine depletion induces changes in morphogenesis and gene expression in the developing cortex

The mesocorticolimbic dopamine (DA) system is implicated in mental health disorders affecting attention, impulse inhibition and other cognitive functions. It has also been involved in the regulation of cortical morphogenesis. The present study uses focal injections of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle of BALB/c mice to examine morphological, behavioral and transcriptional responses to selective DA deficit in the fronto-parietal cortex. Mice that received injections of 6-OHDA on postnatal day 1 (PND1) showed reduction in DA levels in their cortices at PND7. Histological analysis at PND120 revealed increased fronto-cortical width, but decreased width of somatosensory parietal cortex. Open field object recognition suggested impaired response inhibition in adult mice after 6-OHDA treatment. Transcriptional analyses using 17K mouse microarrays showed that such lesions caused up-regulation of 100 genes in the cortex at PND7. Notably, among these genes are Sema3A which plays a repulsive role in axonal guidance, RhoD which inhibits dendritic growth and tubulin beta-5 microtubule subunit. In contrast, 127 genes were down-regulated, including CCT-epsilon and CCT-zeta that play roles in actin and tubulin folding. Thus, neonatal DA depletion affects transcripts involved in control of cytoskeletal formation and pathway finding, instrumental for normal differentiation and synaptogenesis. The observed gene expression changes are consistent with histological cortical and behavioral impairments in the adult mice treated with 6-OHDA on PND1. Our results point towards specific molecular targets that might be involved in disease process mediated by altered developmental DA regulation.

An open-access long oligonucleotide microarray resource for analysis of the human and mouse transcriptomes

Two collections of oligonucleotides have been designed for preparing pangenomic human and mouse microarrays. A total of 148 993 and 121 703 oligonucleotides were designed against human and mouse transcripts. Quality scores were created in order to select 25 342 human and 24 109 mouse oligonucleotides. They correspond to: (i) a BLAST-specificity score; (ii) the number of expressed sequence tags matching each probe; (iii) the distance to the 3′ end of the target mRNA. Scores were also used to compare in silico the two microarrays with commercial microarrays. The sets described here, called RNG/MRC collections, appear at least as specific and sensitive as those from the commercial platforms. The RNG/MRC collections have now been used by an Anglo-French consortium to distribute more than 3500 microarrays to the academic community. Ad hoc identification of tissue-specific transcripts and a ∼80% correlation with hybridizations performed on Affymetrix GeneChip™ suggest that the RNG/MRC microarrays perform well. This work provides a comprehensive open resource for investigators working on human and mouse transcriptomes, as well as a generic method to generate new microarray collections in other organisms. All information related to these probes, as well as additional information about commercial microarrays have been stored in a freely-accessible database called MEDIANTE.