Gene expression analysis in the lungs of Fra-2 Transgenic mice
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ABSTRACT: The aim of this experiment is to identify novel gene candidates being regulated in Fra-2 overexpressing mice. Fra-2 over-expressing mice (Fra-2 TG) develop vascular remodeling and pulmonary fibrosis. Fra-2, as a trascription factor, is responsible for downstream target genes expression. Comparison of the mRNA expressed from WT and Fra-2 TG animals will help to identify Fra-2 target genes. The microarray analysis will be performed in mRNA isolated from lung homogenate. The lungs of the mice were extensively perfused. The right part was snap-froen and transfered to (-80°C). The total RNA was isolated and quantified. Comparison of transgenes vs. wild-types. Hybridization of samples from 4 wild-type and 4 transgene animals = 8 single-color hybridizations
Project description:The aim of this experiment is to identify novel gene candidates being regulated in Fra-2 overexpressing mice. Fra-2 over-expressing mice (Fra-2 TG) develop vascular remodeling and pulmonary fibrosis. Fra-2, as a trascription factor, is responsible for downstream target genes expression. Comparison of the mRNA expressed from WT and Fra-2 TG animals will help to identify Fra-2 target genes. The microarray analysis will be performed in mRNA isolated from lung homogenate. The lungs of the mice were extensively perfused. The right part was snap-froen and transfered to (-80°C). The total RNA was isolated and quantified.
Project description:Microarray analysis of Fra-2 tg female and male mice at 8 weeks of their disease development give an indication of possible differentially regulated genes. Wildtype mice serve as a healthy control.The mRNA was obtained from lung homogenate of the mice (n=4/group for all). There was no treatment applied, we investigate the baseline differences.
Project description:We hypothesized that gene expression in lungs of Fra-1+/+ and Fra-1-/- mice are divergent thus contributing fibrosis. More specifically, Fra-1-/- mice are increased susceptible to fibrosis. In order to test these hypotheses at the gene expression level, we utilized microarray analysis to examine transcriptional differences between Fra-1+/+ and Fra-1-/- mice at early time point.
Project description:We hypothesized that gene expression in lungs of Fra-1+/+ and Fra-1-/- mice are divergent thus contributing fibrosis. More specifically, Fra-1-/- mice are increased susceptible to fibrosis. In order to test these hypotheses at the gene expression level, we utilized microarray analysis to examine transcriptional differences between Fra-1+/+ and Fra-1-/- mice at early time point. This study utilizes microarray analysis to test these hypotheses. 5 days PBS and bleomycin treated lung samples from both Fra-1+/+ and Fra-1-/- mice were used. RNA was isolated and used for global gene expression profiling (Affymetrix MoGene 1.0ST v1 Array). The raw probe signal intensities were quantile normalized over all samples, summarized with the robust multi-array average (RMA) algorithm and log2 transformed with a median polish, using the Affymetrix Power Tools. We considered a transcript cluster (gene-level) to be reliably expressed in a sample if the Affymetrix implemented DABG (detection above ground) p-value was less than 0.05. We used local-pooled-error (LPE) estimates and robust statistical tests for evaluating significance of each gene's differential expression in a comparison.
Project description:Background and Aim: Fra-1 (Fos-related antigen-1) is a member of the AP1 (activator protein-1) family of transcription factors. We have recently shown that Fra-1 is necessary for breast cancer cells to metastasize in vivo, and that breast cancer outcome can be predicted by a classifier comprising genes that are expressed in a Fra-1-dependent fashion. Here, we show that Fra-1 plays an important role also in colon cancer progression. Methods: We compared proliferation rates of parental and Fra-1-depleted colon cancer cells in vitro under 2D, 3D, and attachment-free conditions and in vivo upon subcutaneous and intravenous injections into mice. We also compared RNA expression profiles of colon cancer cells with and without Fra-1 expression. Results: Fra-1 depletion impair colony outgrowth of human colon cancer cells in soft agar and in suspension, whereas it does not affect proliferation on 2D culture plates. Consistent with this, upon subcutaneous injection into mice, tumors formed by Fra-1-depleted colon cancer cells are only three times smaller than those produced by control cells. In contrast, when injected intravenously, Fra-1 depletion causes 200-fold reduction in tumor burden. Consistent with the more aggressive characteristics of Fra-1-proficient tumors, the prognosis of colon cancer patients can be predicted by a Fra-1 classifier generated by comparing RNA profiles of parental and Fra-1-depleted colon cancer cells. Conclusions: Our results demonstrate that Fra-1 is an important determinant of the metastatic potential of human colon cancer cells, and suggest that a Fra-1 classifier can be used as a prognostic predictor in colon cancer patients.
Project description:In the paper "Fra-1 regulates its target genes via binding to remote enhancers without exerting major control on chromatin architecture in triple negative breast cancers" by Bejjani et al., we identified Fra-1 and/or Fra-2 target genes in MDA-MB-231 cells. si RNA against Fra-1 and against Fra-2 were transfected in MDA-MB-231 cells either independenlty or simultaneously to identify genes regulated specifically by Fra-1 or Fra-2 and genes regulated redundantly or complementarily by Fra-1 and Fra-2 total RNA were purified and biotinylated sense-strand cDNA were produced. cDNA targets were used to probe Affymetrix GeneChip Human Gene 2.0 ST arrays
Project description:The polarization and activation of macrophages are controlled synergistically by transcription factors such as NF-κB and AP-1 transcription factor members. Surprisingly, little is known about the role of the Fra proteins, both members of the AP-1 transcription factor family, in macrophage activity. To determine the full profile of Fra network, microarray RNA expression analysis using Agilent Technologies platform was performed in wild-type, Fra-1ΔMxCre or Fra-2ΔLysMCre macrophages.
Project description:Fra-2tg mice develop spontaneous lung fibrosis. We carried out this sequencing with tissue from young animals before they develop the tissue fibrosis, to explore the early gene expression changes that might contribute to the phenotype.