Project description:Analysis of the cystic fibrosis gene Cftr in the colon and small intestine of Cftr-deficient murine model. The hypothesis was loss of Cftr altered expression of genes important in intestinal homeostasis and oncogenic signaling pathways. The results identified potential roles of Cftr in up- or down-regulating major gene clusters that belong to groups of immune response, ion channel, intestinal stem cell and other growth regulators. Total RNA was isolated from the normal intestine of three Apc wildtype Cftr wildtype and three Apc Cftr-deficient mice. For the colon intestinal epithelia from the same region of the distal colon of each mouse was separated from the rest of the intestine prior to RNA isolation. Therefore RNA was obtained from only epithelial cells. For the small intestine, a section of the mid-duodenum from each mouse was sheared of villi prior to RNA isolation. Therefore RNA was obtained from whole duodenum (minus villi), containing epithelia cells but also stromal and other cells. RNA Seq was then conducted on all samples, with at least two replicates for each biological sample.
Project description:De- and re-differentiation represent phenotypic transitions that may contribute to loss of function in ageing musculoskeletal tissues. Applying a systems biology network analysis approach to global gene expression profiles derived from common in vitro culture systems (monolayer and three-dimensional cultures) this study demonstrates common regulatory mechanisms governing de- and re-differentiation transitions in cartilage and tendon cells. Furthermore, evidence of convergence of gene expression profiles during monolayer expansion, and the expression of key developmental markers, challenges the physiological relevance of this culture system.
Project description:Down syndrome (DS) is the most frequent cause of human congenital mental retardation. Cognitive deficits in DS result from perturbations of normal cellular processes both during development and in adult tissues, but the mechanisms underlying DS etiology remain poorly understood. To assess the ability of iPSCs to model DS phenotypes, as a prototypical complex human disease, we generated bona-fide DS and wild-type (WT) non-viral iPSCs by episomal reprogramming. DS iPSCs selectively overexpressed chromosome 21 genes, consistent with gene dosage, which was associated with deregulation of thousands of genes throughout the genome. DS and WT iPSCs were neurally converted at >95% efficiency, and had remarkably similar lineage potency, differentiation kinetics, proliferation and axon extension at early time points. However, at later time points DS cultures showed a two-fold bias towards glial lineages. Moreover, DS neural cultures were up to two times more sensitive to oxidative stress induced apoptosis, and this could be prevented by the anti-oxidant N-acetylcysteine. Our results reveal a striking complexity in the genetic alterations caused by trisomy-21 that are likely to underlie DS developmental phenotypes, and indicate a central role for defective early glial development in establishing developmental defects in DS brains. Furthermore, oxidative stress sensitivity is likely to contribute to the accelerated neurodegeneration seen in DS, and we provide proof of concept for screening corrective therapeutics using DS iPSCs and their derivatives. Non-viral DS iPSCs can therefore recapitulate features of complex human disease in vitro, and provide a renewable and ethically unencumbered discovery platform. Control (WT) and Down Syndrome iPSC lines were generated via episomal reprogramming of human donor fibroblasts. Two iPSC clones conforming to iPS criteria (determined by immunocytochemistry detection of pluripotency markers) were developed from each fibroblast donor. Two control (WT) and DS lines each were further characterized and underwent neural differentiation. Multiple biological replicates of donor fibroblast and iPSC from both control (WT) and DS lines, including 3 euploid DS samples and 3 MEL1 hESC controls (total 54 samples) were hybridized to Illumina HT-12 v4 microarray for gene expression analysis.
Project description:Background: the human placenta facilitates the exchange of nutrients, gas and waste between the fetal and maternal circulations. It also protects the fetus from the maternal immune response. Due to its role at the feto-maternal interface, the placenta is subject to many environmental exposures that can potentially alter its epigenetic profile. Previous studies have reported gene expression differences in placenta over gestation, as well as inter-individual variation in expression of some genes. However, the factors contributing to this variation in gene expression remain poorly understood. Results: in this study, we performed a genome-wide DNA methylation analysis of gene promoters in placenta tissue from three pregnancy trimesters. We identified large-scale differences in global DNA methylation levels between first, second and third trimesters, with an overall progressive increase in average methylation from first to third trimester. The most differentially methylated genes included many immune regulators, reflecting the change in placental immuno-modulation as pregnancy progresses. We also detected increased inter-individual variation in the third trimester relative to first and second, supporting an accumulation of environmentally induced (or stochastic) changes in DNA methylation pattern. These highly variable genes were enriched for those involved in amino acid and other metabolic pathways, potentially reflecting the adaptation of the human placenta to different environments. Conclusions : the identification of cellular pathways subject to drift in response to environmental influences provide a basis for future studies examining the role of specific environmental factors on DNA methylation pattern and placenta-associated adverse pregnancy outcomes. A total of 42 samples, with 18 first trimester, 10 second trimester, 14 full term placenta
Project description:Genome-wide expression profiling of stably NGFR transfected melanoma cells was used to identify genes driven by expression of the nerve growth factor receptor CD271 (NGFR). Stable overexpression of NGFR (CD271): Generation of cell lines stably overexpressing CD271 (NGFR), melanoma cells were transfected with 2 µg of a plasmid expressing GFP-tagged human NGFR (RG207966, OriGene) and selected with G418 (100-300 µg/ml, PAA) over a period of two weeks followed by sub-cloning or FACS. Gene expression profiling: Whole genome expression profiling of T20/02 and A375 cells (NGFR) and control cells (Mock or GFP) was performed with three biological replicates. Illumina raw data of BeadChip HumanHT-12V4 platform were summarized via the BeadStudio without normalization and background correction. Follow-up processing was done via the R/Bioconductor environment employing packages lumi, limma and q-value. Data were normalized with quantile normalization. Genes were termed significantly differentially expressed when the average detection p-value of at least one case was < 0.05 the ratio was outside the interval [0.75,1.33], one of the p-values from limma test, Student's t-test, Welch test and Wilcoxon test was < 0.05. At least one of the q-values corresponding to one of these tests < 0.05.
Project description:One major class of anti-cancer drugs targets topoisomerase II to induce DNA double-strand breaks and cell death of fast growing cells. In vitro experiments showed that doxorubicin can induce histone eviction as well as DNA damage, while etoposide can only induce DNA damage. Here, we compare the transcription responses of different tissues to doxorubicin or etoposide treatment in vivo. Total RNA from respective tissues in FVB mice 1 day or 6 days post indicated drug treatment were extracted and compared to un-treated mice. Two mice were used for each treatment.
Project description:One major class of anti-cancer drugs targets topoisomerase II to induce DNA double-strand breaks and cell death of fast growing cells. Here, we compare three members of this class - the antracyclines doxorubicin and aclarubicin, and a chemically unrelated compound, etoposide. Aclarubicin does not induce DNA breaks. We define a new activity for the antracyclines: unsupported histone eviction from ´open´ or loosely packed chromosomal areas reflecting exon and promoter regions. As a result, the epigenome and the transcriptome are strongly affected. Tissue culture cells were treated with doxorubicin, aclarubicin or etoposide for 2 hours. Then drugs were removed by extensive washing. cells were further cultured for indicated days before total RNA were extracted and compared to un-treated control.
Project description:One major class of anti-cancer drugs targets topoisomerase II to induce DNA double-strand breaks and cell death of fast growing cells. Here, we compare three members of this class - the antracyclines doxorubicin and aclarubicin, and a chemically unrelated compound, etoposide. Aclarubicin does not induce DNA breaks. We define a new activity for the antracyclines: unsupported histone eviction from ´open´ or loosely packed chromosomal areas reflecting exon and promoter regions. As a result, the epigenome and the transcriptome are strongly affected. Tissue culture cells were treated with doxorubicin, aclarubicin or etoposide for 2 hours. Then drugs were removed by extensive washing. cells were further cultured for indicated days before total RNA were extracted and compared to un-treated control.