Expression data from three rice lines (1-control, 1-transgenic and 1-negative segregant) throughout generations and under salt stress
Ontology highlight
ABSTRACT: The approval of genetically modified (GM) crops is preceded by years of intensive research to demonstrate safety to humans and environment. We recently showed that in vitro culture stress is the major factor influencing proteomic differences of GM vs. non-GM plants. This made us question the number of generations needed to erase such memory. We also wondered about the relevance of alterations promoted by transgenesis as compared to environment-induced ones. Here we followed three rice lines (1-control- C, 1-transgenic- Ta and 1-negative segregant- NSb) throughout eight generations after transgenesis, and further analyzed their response to salinity stress on the F6 generation. Three pools of 10 whole fifteen days-old rice seedlings (Oryza sativa L. ssp. japonica cv. Nipponbare) were selected from each line at F4, F6 and F8 generations. Because salinity stress was imposed on half of the seedlings (C, Ta and NSb) in F6 generation, from this generation onwards we worked with six rice lines (C, Csalt, Ta, Tasalt, NSb, NSbsalt).
Project description:The approval of genetically modified (GM) crops is preceded by years of intensive research to demonstrate safety to humans and environment. We recently showed that in vitro culture stress is the major factor influencing proteomic differences of GM vs. non-GM plants. This made us question the number of generations needed to erase such memory. We also wondered about the relevance of alterations promoted by transgenesis as compared to environment-induced ones. Here we followed three rice lines (1-control- C, 1-transgenic- Ta and 1-negative segregant- NSb) throughout eight generations after transgenesis, and further analyzed their response to salinity stress on the F6 generation.
Project description:Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer (10.7554/eLife.03848). Specifically, the 2.7 kb Mouse Smarcd3-F6 sequence (chr5: 24113559 -24116342 from mm9 assembly) was sub-cloned from a gateway entry vector into the Zebrafish Enhancer Detection (ZED). Tol2-mediated transgenesis was performed and stable lines were created. The Smarcd3-F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3-F6:EGFPhsc70 embryos were dissociated at 10 hours post fertilization for fluorescent activated cell sorting (FACS). 30,000-50,000 GFP positive and negative cells were collected from FACS for ATAC-seq. ATAC-seq libraries were created with Tn5 transposase and single-end sequenced on the Illumina HiSeq 2500 platform
Project description:F6 murine leukaemic cells were transfected either with MHC II or with GFP and transcriptionally profiled by RNA-seq. Triplicate samples for each genotype were sequenced. This is part of ongoing investigation of a possible cell-intrinsic role of MHC II B cells.
Project description:Stable zebrafish cell lines were created that expressed GFP under the control of a previously characterized mouse Smarcd3 enhancer (10.7554/eLife.03848). Specifically, the 2.7 kb Mouse Smarcd3-F6 sequence (chr5: 24113559 -24116342 from mm9 assembly) was sub-cloned from a gateway entry vector into the Zebrafish Enhancer Detection (ZED). Tol2-mediated transgenesis was performed and stable lines were created. The Smarcd3-F6:EGFPhsc70 allele was used for all genomics experiments. Smarcd3-F6:EGFPhsc70 embryos were dissociated at 10 hours post fertilization for fluorescent activated cell sorting (FACS) to collect GFP+ cells. Single-cell cDNA libraries were prepared using Fluidgim C1 system ((Fluidgim, PN 100-7168 Rev. B1)). 96 single-cell libraries were collected from three batches of experiments and sequenced on the Illumina HiSeq 2500 platform. Data from 92 cells (number of genes detected > 2000) were kept for clustering analysis. For the tube control experiments we performed mRNA-seq on ~4000 cells from the same embryo batches as the scRNA-seq experiments. GFP negative cells were also included to enable a GFP+ versus GFP- comparison. Libraries were made following the Fluidgim C1 mRNA-seq tube control protocol (Fluidgim, PN 100-7168 Rev. B1). Polyadenylated mRNA was captured by Oligo-dT primers and PCR amplified after reverse transcription. Final sequencing libraries were made using Nextera XT DNA Sample Preparation Kit and pair-end sequenced on the Illumina HiSeq 2500 platform.
Project description:The SnRK1 protein kinase, the plant ortholog of mammalian AMPK and yeast Snf1, is activated by the energy depletion caused by adverse environmental conditions. Upon activation, SnRK1 triggers extensive transcriptional changes to restore homeostasis and promote stress tolerance and survival partly through the inhibition of anabolism and the activation of catabolism. Despite the identification of a few bZIP transcription factors as downstream effectors, the mechanisms underlying gene regulation, and in particular gene repression by SnRK1, remain mostly unknown. microRNAs (miRNAs) are 20-24nt RNAs that regulate gene expression post-transcriptionally by driving the cleavage and/or translation attenuation of complementary mRNA targets. In addition to the well-established role of miRNAs as regulators of plant development, mounting evidence implicates miRNAs in the response to environmental stress. Given the involvement of miRNAs in stress responses and the fact that some of the SnRK1-regulated genes are miRNA targets, we postulated that miRNAs drive part of the transcriptional reprogramming triggered by SnRK1 activation. To test this we have performed comparative analyses of the transcriptional response to energy deprivation between WT and dcl1-9, a mutant deficient in miRNA biogenesis. To assess the impact of miRNA deficiency on the starvation response we performed transcriptomics analyses of WT and dcl1-9 plants by subjecting leaves to 6h of light (control) or darkness (starvation)
Project description:The limited efficacy of currently approved immunotherapies in EGFR-mutant lung adenocarcinoma (LUAD) underscores the need to better understand alternative mechanisms governing local immunosuppression to fuel novel therapies. Elevated surfactant and GM-CSF secretion from the transformed epithelium induces tumor-associated alveolar macrophages (TA-AM) to proliferate and support tumor growth by rewiring inflammatory functions and lipid metabolism. TA-AM properties are driven by increased GM-CSF—PPARγ signaling and inhibition of airway GM-CSF or PPARγ in TA-AMs suppresses cholesterol efflux to tumor cells, which impairs EGFR phosphorylation and restrains LUAD progression. In the absence of TA-AM metabolic support, LUAD cells compensate by increasing cholesterol synthesis, and blocking PPARγ in TA-AMs simultaneous with statin therapy further suppresses tumor progression and increases T cell effector functions. These results reveal new therapeutic combinations for immunotherapy resistant EGFR-mutant LUADs and demonstrate how such cancer cells can metabolically co-opt TA-AMs through GM-CSF—PPARγ signaling to provide nutrients that promote oncogenic signaling and growth.
Project description:The murine thymus produces discrete γδ T cell subsets making either IFN-γ or IL-17, but the role of the TCR in this developmental process remains controversial. Here we generated a non-transgenic and polyclonal model of reduced TCR expression and signal strength selectively on γδ T cells. Mice haploinsufficient for both CD3γ and CD3δ (CD3DH) showed normal αβ thymocyte subsets but specific defects in γδ T cell development, namely impaired differentiation of IL-17-producing embryonic Vγ6+ (but not adult Vγ4+) γδ T cells and a marked depletion of IFN-γ-producing CD122+ NK1.1+ (Vγ1-biased) γδ T cells throughout life. As result, adult CD3DH mice showed defective peripheral IFN-γ responses and were resistant to experimental cerebral malaria. Thus, strong TCR signaling is required within specific developmental windows with distinct Vγ usage and differential cytokine production by effector γδ T cell subsets. We investigated the transcriptional changes associated with reduced TCRγδ signaling in the CD3DH model. Transcriptome-wide analysis of FACS-purified CD3DH or WT γδ thymocytes from E18 or 6-week was carried looking for patterns of gene expression during ontogeny
Project description:The biological effects of TTR proteins in the vasculature remain unknown. We used microarrays to detail the modulation of gene expression on HUVECs by V30M TTR when compared to cells exposed to WT TTR. HUVECs (passage 7) were cultured in the presence of WT or V30M TTR at 4M-BM-5M for 3 hours. RNA was extracted and hybridized on Affymetrix microarrays. We sought to obtain differentially expressed genes modulated by V30M TTR protein.
Project description:Coupling immunity and development is essential to ensure survival despite changing internal conditions in the organism. The metamorphosis of the fruit fly represents a striking example of drastic and systemic physiological changes that need to be integrated with the innate immune system. However, the mechanisms that coordinate development and immune cell activity in the transition from larva to adult in Drosophila remain to elucidate. The steroid hormone ecdysone is known to act as a key coordinator of metamorphosis. This hormone activates a nuclear receptor, the Ecdysone Receptor (EcR), which acts as a heterodimer with its partner Ultraspiracle (USP). Together, they activate the transcription of primary response genes, which in turn activate the transcription of a battery of late response genes. We have revealed that regulation of macrophage-like cells (hemocytes) by the steroid hormone ecdysone is essential for an effective innate immune response over metamorphosis. We have shown that in response to ecdysone signalling, hemocytes rapidly up regulate actin dynamics, motility and phagocytosis of apoptotic corpses, and acquire the ability to chemotax to damaged epithelia. Most importantly, individuals lacking ecdysone-activated hemocytes are defective in bacterial phagocytosis and are fatally susceptible to infection by bacteria ingested at larval stages, despite the normal systemic production of antimicrobial peptides. This decrease in survival is comparable to the one observed in pupae lacking immune cells altogether, indicating that ecdysone-regulation is essential to hemocyte immune functions and survival after infection. To better understand the ecdysone regulation of hemocyte activities, we have performed gene expression analysis. In order to identify the genes which expression change at the onset of metamorphosis, we have sorted hemocytes from 3rd instar larvae and from young prepupae and compared their gene expression. Moreover, and in order to identify which genes are regulated by the ecdysone signalling, we have used individuals expressing a dominant negative form of the Ecdysone Receptor specifically in their hemocytes. We have sorted hemocytes from 3rd instar and young prepupae of this genotype to compare their gene expression to the gene expression in larvae and prepupae from the control individuals. Hemocytes were isolated by FACS from selected 3rd instar larvae (at the late feeding stage) and prepupae (from 1h to 2h after puparium formation - APF) corresponding to two different genotypes: individuals w;HmlDeltaGal4, UAS-GFP/+ that express GFP specifically in hemocytes (genotype control), and individuals w;HmlDeltaGal4; UAS-GFP/UAS-EcRB1DN W650A which hemocytes express an Ecdysone Receptor Dominant Negative construct in addition to the GFP (EcRDN). For each of the four conditions we performed three biological replicates.
Project description:Attempts to establish a tissue bank from autopsy samples have led to uncovering of the secrets of many diseases. Here, we examined the length of time that the RNA from postmortem tissues is available for microarray analysis and reported the gene expression profile for up- and down-regulated genes during the postmortem interval (PMI). We extracted RNA from fresh-frozen (FF) and formalin-fixed paraffin-embedded (FFPE) brains and livers of three different groups of mice: 1) mice immediately after death, 2) mice that were stored at room temperature for 3 h after death, and 3) mice that were stored at 4°C for 18 h after death, as this storage resembles the human autopsy process in Japan. Based on the microarray analysis, we selected genes that were altered by >1.3-fold or <0.77-fold and classified these genes using hierarchical cluster analysis following DAVID (database for annotation, visualization, and integrated discovery) gene ontology analysis. These studies revealed that cytoskeleton-related genes were enriched in the set of up-regulated genes, while serine protease inhibitors were enriched in the set of down-regulated genes. Interestingly, although the RNA quality was maintained, up-regulated genes were not validated by quantitative PCR, suggesting that these genes may become fragmented or modified by an unknown mechanism. We extracted RNA from fresh-frozen (FF) brains and livers from mice under three different conditions: 1) mice just after death as a control, 2) mice that were stored at room temperature for 3 h after death, and 3) mice that were stored at 4°C for 18 h after death to resemble the human autopsy process. We also created formalin-fixed paraffin-embedded (FFPE) tissue blocks at the same time using mouse organs obtained under the three conditions described above. We then purified RNA from the FFPE tissue blocks. Furthermore, we performed microarray analysis to examine changes in the gene expression profiles during the postmortem interval in FF samples and to compare ene expression profiles between FF and FFPE samples at three different postmortem times as described for the FF samples.