Project description:With regulatory roles in development, cell proliferation and disease, micro-RNA (miRNA) biology is of great importance and a potential key to novel RNA-based therapeutic regimens. Biochemically based sequencing approaches have provided robust means of uncovering miRNA binding landscapes on transcriptomes of various species. However, a current limitation to the therapeutic potential of miRNA biology in cattle is the lack of validated miRNAs targets. Here, we use cross-linking immunoprecipitation (CLIP) of the Argonaute (AGO) proteins and unambiguous miRNA-target identification through RNA chimeras to define a regulatory map of miRNA interactions in the cow (Bos taurus). The resulting interactome is the deepest reported to date for any species, demonstrating that comprehensive maps can be empirically obtained. We observe that bovine miRNA targeting principles are consistent with those observed in other mammals. Motif and structural analyses define expanded pairing rules with most interactions combining seed-based pairing with distinct, miRNA-specific patterns of auxiliary pairing. Further, miRNA-target chimeras had predictive value in evaluating true regulatory sites of the miR-17 family. Finally, we define miRNA-specific targeting for >5000 mRNAs and determine gene ontologies (GO) for these targets. This confirmed repression of genes important for embryonic development and cell cycle progress by the let-7 family, and repression of those involved in cell cycle arrest by the miR-17 family, but it also suggested a number of unappreciated miRNA functions. Our results provide a significant resource for transcriptomic understanding of bovine miRNA regulation, and demonstrate the power of experimental methods for establishing comprehensive interaction maps. Overall design: miRNA-target chimeras were identified in MDBK cells from standard AGO-CLIP or CLEAR-CLIP experiments as indicated. Experiments were included where cells had been exposed to infection with bovine viral diarrhea virus or tinyLNA-17 treatment. These are indicated. Processed reads were aligned to the host genome (BosTau7). Alignment statistics of CLIP reads are given in the processed data file "Table S1". A complete table of significant peaks from standard AGO-CLIP reads of the 39 data sets is given in "Table S2". This table additionally contains information regarding the associated CLIP read cluster (clustering of all CLIP reads) and potential overlapping miRNA-target chimeras, as well as genomic annotation. A complete list of individual miRNA-target chimeras is given in "Table S3". In the "RNA-seq: tinyLNA-17 vs. Mock" subseries, mRNA-seq was performed on two replicates each of MDBK cells treated with the miR-17 family inhibitor, tinyLNA-17. Differential gene expression analysis was performed and the associated data is given as processed data file "Table S5".
Project description:Affymetrix Human GeneChips are used to profile gene expression of bovine tissues and embryos to identify uniquely expressed genes in bovine in-vitro fertilized embryos by comparing with seven bovine adult tissues through gene clustering
Project description:Bene expression profile of Angus bovine testis tissue at 2, 4 and 8 weeks of age using Affymetrix Bovine GeneChip Experiment Overall Design: Samples obtained from Angus bull calves at 2, 4 and 8 weeks of age. Two replicates at each age, 6 total samples.
Project description:The liver of dairy cows naturally displays a series of metabolic adaptation during the periparturient period in response to the increasing nutrient requirement of lactation. The hepatic adaptation is partly regulated by insulin resistance and it is affected by the prepartal energy intake level of cows. We aimed to investigate the metabolic changes in the liver of dairy cows during the periparturient at gene expression level and to study the effect of prepartal energy level on the metabolic adaptation at gene expression level.B13:N13 Overall design: Sixteen Ayrshire dairy cows were allocated to a controlled-energy diet (CON) or a high-energy diet (HIGH) for 6 weeks before the predicted parturition. Microarrary was conducted for liver samples at 8 d prior to the predicted parturition (-8 d) and 1 d and 9 d after the actual parturition (1 d and 9 d).
Project description:Yamoa™ is marketed and sold as a dietary supplement with anecdotal positive effects in asthma and hay fever. We determined that Yamoa™ (ground bark of Funtumia elastica tree) stimulated innate immunity in part by affecting gamma delta T cells. Yamoa™ had distinct priming effects, very similar to, but more robust than, that of lipopolysaccharide (LPS), on bovine, mouse and human gamma delta T cells. However, the optimal effect was dependent on the presence of accessory cells. Gene expression patterns in bovine gamma delta T cells and monocytes induced by Yamoa™ were very similar to those induced by ultrapure LPS, but the agonists in Yamoa™ did not signal entirely through TLR4. Yamoa™ stimulated human cells to produce cytokines involved innate protection. The bioactive component of Yamoa™ was delineated to a complex polysaccharide fraction (Yam-I). Intraperitoneal injection of Yamoa™ and very low doses of Yam-I in mice induced rapid increases peritoneal neutrophils directed by changes chemokine expression. Yamoa™ and Yam-I were effective as therapeutic treatments in mice with Salmonella enterica serotype Typhimurium (ST) induced enterocolitis that resulted in decreased bacterial counts in feces. This initial characterization of the immune stimulatory properties of polysaccharides derived from Yamoa™ suggests potential mechanisms for positive effects in asthma and that they have potential for application in infectious disease settings. . Experiment Overall Design: To begin to understand the effects of Yamoa in innate immunity, we investigated the global gene expression profiles of stimulated bovine gamma delta T cells. Peripheral blood from 3 neonatal bovine calves was collected. gamma delta T cells were sorted to >97% purity using a FACS Vantage. Cells were placed in culture and stimulated with either an aqueous extract of Yamoa (32.6ug/ml), ultrapure LPS [uLPS (10ug/ml)] or PBS for 4 hours after which RNA was extracted and processed for microarray analysis.
Project description:Angiotensin II (Ang-II) regulates adrenal steroid production and gene transcription through several signaling pathways. Changes in gene transcription occur within minutes after Ang-II stimulation, causing an acute increase in aldosterone production and subsequent increase in the overall capacity to produce aldosterone. Our goal was to compare the Ang-II regulation of early gene expression and confirm the upregulation of selected genes using quantitative real-time RT-PCR (qPCR) across three species: human, bovine, and rat. Experiment Overall Design: Microarray analysis was performed using samples from control and Ang-II-(10 nM) treated (1 hour) cells from human adrenocortical tumor cell line H295-R, and primary adrenal glomerulosa cells from bovine and rat, applied respectively to human HG-133 + 2 , bovine, and rat 230-2 Affymetrix chips. qPCR was performed to confirm upregulation of selected genes using mRNA. Dye Swap was not used. Human samples (H295R) include 3 replicates of Basal (controls) and 3 repeats of Angiotensin II samples. Human H295R cells include also a group of cycloheximide (protein synthesis blocker) and angiontensin II + cycloheximide in order to check if the genes were direct targets of angiotensin II.