Transcription profiling of Arabidopsis wild type, MYB46 over-expressing, MYB83 over-expressing and myb46/myb83 double mutants to investigate the regulation mechanism of secondary cell wall synthesis during xylem vessel differentiation
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ABSTRACT: To investigate the regulation mechanism of secondary cell wall synthesis during xylem vessel differentiation, we analyzed the function of two Arabidopsis MYB transcription factors, MYB46 and MYB83. Microarray analysis was performed for 11-day old seedlings of wild-type, MYB46 over-expressor, MYB83 over-expressor, and myb46 myb83 double mutant using Affymetrix ATH-1 Genome Array. Gene expressions in MYB46 over-expressor, MYB83 over-expressor, and myb46 myb83 double mutant were compared to those in wild-type.
Project description:Multiple-condition experiment was desinged to be any number of conditions in an experiment without replicate observations for microarray and used to identify genes differentially expressed between different pairs of conditions (treatments).<br> In this study we used breast cancer stable cell lines for overexpressing and silencing annexin A1 (ANXA1), which belongs to a family of -dependent phospholipid binding proteins and are preferentially located on the cytosolic face of the plasma membrane. Cell lines overexpressing ANXA1 (MDA_MB-453/cDNA) were generated by introducing retroviral vectors containing ANXA1 cDNA (pBabe/ANXA1 cDNA) into breast cancer cell line MDA-MB-453 (a low expressor of ANXA1). Breast cancer cell line BT-474, a high expressor of ANXA1, was infected with ANXA1 siRNA-plasmid viruses to knockdown ANAXAI expressor (BT-474/siRNA) where nucleotides corresponding to siRNA were synthesized and ligated into the pLNCX retroviral vector [35,36]. We also used a pLNCX/U6 empty vector to infect BT-474 and obtained an empty vector expressor. Therefore, 5 breast cancer cell lines (MDA_MB-453, MDA_MB-453/cDNA, BT-474, BT-474/siRNA, and BT-474/U6) are attributed to two genotypes: MDA_MB-453 and BT-474. MCE was performed for microarray analysis with these 5 breast cancer cell lines, that is, only one sample was drawn from each breast cancer cell line.
Project description:The HMGA2 gene is frequently deranged by translocation and/or amplification in mesenchymal tumours, generally leading to over-expression of shorted transcripts and a truncated protein. In this experiment we have over-expressed wild type and truncated HMGA2 protein in an immortalized mesenchymal stem-like cell line and investigated their effects on gene expression patterns.
Project description:Activation of plant immunity by bacterial MAMPs such as flg22 affects nucleosome positioning over thousands of loci and is correlated with the transcription of immune-related genes. Data for chromatin remodelling ATPase EDA16 truncated and over-expressor mutants as well as Col-0 (control) are provided.
Project description:In order to identify candidate target genes of the OBP1 (At3g50410) transcription factor we used dexamethasone inducible system (Lloyd et al, 1994). A single inducible over-expression line was compared to an empty vector control line 10h after DEX induction to identify candidate genes that were confirmed by quantitative RT-PCR.
Project description:In human cells, Staufen1 is double-stranded RNA-binding protein involved in several cellular functions including mRNA localization, translation and decay. We used a genome wide approach to identify and compare the mRNA targets of mammalian Staufen1. The mRNA content of Staufen1 mRNPs was identified by probing DNA microarrays with probes derived from mRNAs isolated from immunopurified Staufen-containing complexes following transfection of HEK293T cells with a Stau1-HA expressor. Our results indicate that 7% of the cellular RNAs expressed in HEK293T cells are found in Stau1-containing mRNPs. There is a predominance of mRNAs involved in cell metabolism, transport, transcription, regulation of cell processes and catalytic activity. Experiment Overall Design: HEK293 cells were transiantly transfected with plasmids coding for Staufen1-HA. Cell extracts were immunoprecipitated using anti-HA antibodies and co-immunoprecipitated mRNAs were purified and used to hybridize microarrays. As control, cell extract from mock transfected cells were used.
Project description:Transient genetic modification of plant protoplasts is a straightforward and rapid technique for the analysis of numerous aspects of plant biology. One drawback in the analysis of transformed protoplast suspensions is that they are a heterogeneous mix of cells that have and have not been successfully transfected. To overcome this problem, we have developed a system that employs a fluorescent positive selection marker in combination with flow cytometric analysis as well as fluorescence activated cell sorting (FACS) to isolate responses in the transfected protoplasts exclusively. This recombinase-compatible system enables high-throughput screening of genetic circuitry. Moreover, the use of FACS allows in depth downstream analysis. Lastly, over-expression is an effective means to dissect regulatory networks, especially where redundancy exists. Here, this system has been applied to the study of auxin signaling in order to investigate reporter gene activation and genome-wide transcriptional changes in response to manipulation of the auxin-response network. We have transiently over-expressed dominant negative mutant isoforms of Aux/IAA transcription factors (IAA7mII and IAA19mII; Tiwari et al., 2001) in Arabidopsis Pwer::GFP root protoplasts, making use of a RFP fluorescent positive selection marker and FACS to isolate the dually labeled (IAAnmII expressing and Pwer::GFP-positive) cells. We have compared the transcriptional differences between an empty vector control, IAA7mII and IAA19mII protoplasts that had either been treated with 5microM IAA or mock-treated for 3 hours. Experiment Overall Design: 18 samples with 3 replicates for each condition and transformation vector: 3x empty vector mock treated, 3x empty vector IAA treated, 3x IAA7mII over-expressor mock treated, 3x IAA7mII over-expressor IAA treated, 3x IAA19mII over-expressor mock treated and 3x IAA19mII over-expressor IAA treated.
Project description:MycRas vs Ras colon carcinoma. Human adenocarcinomas commonly harbor mutations in the Ki-Ras and c-Myc proto-oncogenes and the trp53 tumor suppressor gene. All three genetic lesions are potentially pro-angiogenic, since they sustain production of the vascular endothelial growth factor (VEGF). Yet murine Ki-Ras/p53-null colonocytes formed indolent, poorly vascularized tumors, whereas additional transduction with a Myc-encoding retrovirus promoted vigorous vascularization and growth. While VEGF levels were unaffected by Myc, enhanced neovascularization correlated with down-regulation of anti-angiogenic thrombospondin-1 (Tsp1) and related proteins, such as connective tissue growth factor (CTGF). Both Tsp1 and CTGF are predicted targets for repression by the miR-17-92 microRNA cluster, which was upregulated in RasMyc colonocytes. Indeed, miR-17-92 knock-down with antisense 2’-O-methyl oligoribonucleotides partly restored Tsp1 and CTGF expression, and conversely, transduction of Ras-only cells with a miR-17-92-encoding retrovirus reduced Tsp1 and CTGF levels. Importantly, miR-17-92-transduced cells formed larger, better perfused tumors. These findings establish a role for microRNAs in non-cell-autonomous Myc-induced tumor phenotypes.
Project description:In a transgenic mouse model of Alzheimer disease (AD), cleavage of the amyloid precursor protein (APP) by the α-secretase ADAM10 prevented amyloid plaque formation and alleviated cognitive deficits. Furthermore, there was a positive influence of ADAM10 over-expression on neurotransmitter-specific formation of synapses and on synaptic plasticity. To assess the influence of ADAM10 on the gene expression profile in the brain we performed microarray analysis using RNA isolated from brains of five month old mice over-expressing either the α-secretase ADAM10 or a dominant-negative mutant (dn) of this enzyme. As compared to non-transgenic wild-type mice, 355 genes were found to be differentially expressed in ADAM10 transgenic mice and 143 genes in dnADAM10 mice. A higher number of genes was found to be differentially regulated in double-transgenic mouse strains additionally expressing the human APP V717I mutant (APP[V717I]). Thus, α-secretase cleavage of over-expressed APP[V717I] alters CNS gene expression additionally. Keywords: genetic modification