Unknown,Transcriptomics,Genomics,Proteomics

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FDB1_GM-CSFTyr577_Receptor_Mutant_Factorial_Time_Course_Study

ABSTRACT: To understand better the mechanisms controlling the balance between proliferation and and differentiation during myelopoiesis we have utilized teh bi-potent FDB1 myeloid cell line which differentiates to granulocytes and macrophages in response to GM-CSF and thus provides a dissectable model to analyse the switch between growth and differentiation. This was further studied using this cell line in which a second site mutation Y577F had been generated. A factorial time-course design between the two cell lines and over time, combined with linear modelling and geneset enrichment analysis identified the expression changes associated with the switch between granulocyte differentiation and macrophage differentiation. We observed that a single intracellular tyrosine residue (Tyr577) mediates the granulocyte fate decision. The loss of granulocyte differentiation and enhanced macrophage differentiation observed in this second site mutant is associated with accumulation of Beta-Catenin and activation of Tcf4 and other Wnt Target genes. Factorial design. First Factor was cell type with levels FDB1 expressing FI-Delta receptor mutant and FDB1 expressing FI-Delta Y577F receptor mutant. Second factor is time with levels 0 and 72 hours. Two biological replicates wre prepared for each cell line and comparions were perfomed for each cell line and directly between cell lines at 0 and 72hs. Additional dye swaps were done with FI Delta and FIDelta Y577F at 0h and the FI Delta 0-72h comparison.

ORGANISM(S): Mus musculus

SUBMITTER: Chung Kok

PROVIDER: E-GEOD-25857 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Aims of study: (1) To identify systemic differences in osteoarthritic (OA) bone that contribute to OA pathogenesis. (2) Identify novel osteoporotic (OP) bone-related disease genes. Study involved comparison of trabecular bone extracted from the intertrochanteric (IT) region of the proximal femur (PF) from OA, OP and normal/control (CTL) individuals. Bone was obtained from OA and OP individuals at surgery for total hip replacement and from CTL individuals at autopsy. Keywords: Bone tissue comparison, diseased versus non-diseased, factorial design, linear modelling Four sets of sample comparisons (39 comparisons in total) were made in this study. These comprised 10 OA-CTL female, 10 OA-CTL male, 10 OA-OP female and 9 OP-CTL female comparisons. A Compugen Human 19K-oligo library spotted onto glass slides by the Adelaide Microarray facility (AMF) was used in this study. The slides were interrogated by competitive hybridisation of Cy3 and Cy5 labelled pairs of OA-CTL, OA-OP or OP-CTL amplified RNA samples. Sample pairs were age-matched as closely as possible. A biological dye-swap strategy was employed. After hybridisation and washing of the slides they were scanned using a GenePix 4000B Scanner driven by GenePix Pro 4.0. All analyses were performed using the statistical programming and graphics environment R. The “SPOT” software package was used to identify spots by adaptive segmentation method and subtract backgrounds utilising morphological opening approach. Data analysis was performed in R using Bioconductor. Loess print tip method was used to correct for dye-bias and intensity within each group of adjacent spots printed by one pin. Linear modelling was performed with the Limma package of Bioconductor. Linear models were used to incorporate all available data into a single analysis. This allowed the use of both direct and indirect comparisons in calculation of expression ratios as well as improved accuracy in estimation of variance for each gene. Factorial design utilised the following factors: medical condition (OA or OP or CTL); sex (male or female) for OA patients only.

Project description:Microarray technology provides a powerful tool for gene discovery studies, but the development of microarrays for individual species can be expensive and time-consuming. In this study, we test the suitability of a Danio rerio oligonucleotide microarray for application in a species with few genomic resources, the coral reef fish Pomacentrus moluccensis. Coral reef fishes are expected to experience rising sea surface temperatures due to climate change. How well tropical reef fish species will respond to these increased temperatures and which genes are important for resistance and adaptation to elevated temperatures is not known. Microarray technology may help identify candidate genes for thermal stress resistance in coral reef fishes. Results from a comparative genomic DNA hybridisation experiment and direct sequence comparisons indicate that for most genes there is significant sequence similarity between P. moluccensis and D. rerio, suggesting that the D. rerio array is applicable to P. moluccensis. Heterologous microarray experiments on heat-stressed P. moluccensis identified changes in transcript abundance at 120 gene loci, with many genes involved in protein processing, transcription, and cell growth. Changes in transcript abundance for a selection of candidate genes were confirmed by quantitative real-time PCR. We have demonstrated that heterologous microarrays can be successfully employed to study non-model organisms. Such a strategy thus greatly enhances the applicability of microarray technology to the field of environmental and functional genomics and will be useful for investigating the molecular basis of thermal adaptation in coral reef fishes. Keywords: stress response, comparative genomic hybridization (CGH) Common reference design [Stress response_P. moluccensis]: four individual treatment fish (heat-stressed) are contrasted in four microarray hybridisations against a pooled control consisting of four fish kept at ambient temperature. All eight fish employed in this analysis were wild-captured and are biological replicates. The experiment included dye-swap, i.e. stressed fish were labelled red in two hybridisations and green in the other two hybridisations. Common reference design [CGH_P. moluccensis and D. rerio]: four individual P. moluccensis gDNA samples are contrasted in four microarray hybridisations against a pooled gDNA sample consisting of three D. rerio. The experiment included dye-swaps.

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FDB1_GM-CSFTyr577_Receptor_Mutant_Factorial_Time_Course_Study

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