Gene expression of colon from control or VilCre;Ihhflox/flox mice
ABSTRACT: Ihh expression is required for intestinal stem cell niche development. Loss of Ihh leads to disruption of intestinal stem cell niche, which leads to abnormal stem cell development and epithelial cell differentiation. 3 paired control (C) and mutant (M) mice here harvested. Their colon tissues were analyzed using Affymetrix Mouse Gene 1.0ST array Control and VilCre;Ihhflox/flox mice were harvested at p12 There are 3 pairs (total 6) samples: M1 and C1 are littermates M2 and C2 are littermates M3 and C3 are littermates
Project description:Ihh expression is required for intestinal stem cell niche development. Loss of Ihh leads to disruption of intestinal stem cell niche, which leads to abnormal stem cell development and epithelial cell differentiation. Overall design: 3 paired control (C) and mutant (M) mice here harvested. Their colon tissues were analyzed using Affymetrix Mouse Gene 1.0ST array Control and VilCre;Ihhflox/flox mice were harvested at p12 There are 3 pairs (total 6) samples: M1 and C1 are littermates M2 and C2 are littermates M3 and C3 are littermates
Project description:Primordial germ cells (PGCs), the embryonic precursors of eggs and sperm, are a unique model for identifying and studying regulatory mechanisms in singly migrating cells. From their time of specification to eventual colonization of the gonad, mouse PGCs traverse through and interact with many different cell types, including epithelial cells and mesenchymal tissues. Work in drosophila and zebrafish have identified many genes and signaling pathways involved in PGC migration, but little is known about this process in mammals. We have generated a point mutation in the Ror2 gene that we know disrupts primordial germ cell migration in the developing mouse embryo. We used microarray analysis to determine if this defect is mediated through genome-wide or pathway-specific transcriptional changes. We analyzed primordial germ cells (PGCs) from 4 wild-type (WT) and 4 Ror2Y324C/Y324C mutant embryos using Oct4-DPE-EGFP. PGCs were collected during their active migratory state at embryonic day 9.5 (somite range 20-25).
Project description:Tumors that show evidence of epithelial to mesenchymal transition (EMT) have been associated with metastasis, drug resistance, and poor prognosis. EMT may alter the molecular requirements for growth and survival in different contexts, but the underlying mechanisms remain incomplete. Given the heterogeneity along the EMT spectrum between and within tumors it is important to define the requirements for growth and survival in cells with an epithelial or mesenchymal phenotype to maximize therapeutic efficacy. We have established an inducible cell line model in which a tamoxifen regulatable Twist-ER fusion protein is stably expressed in the H358 non-small cell lung cancer cell line. Upon tamoxifen addition, cells undergo EMT and provide a system in which we can compare the growth and survival requirements directly related to EMT, removing confounding factors present when comparing different cell lines. H358 cells stably expressing either GFP or TwistER were treated for 12 days in culture with 100nM 4-hydroxytamoxifen followed by RNA isolation. Three biological replicates of each condition were collected.
Project description:Natural isolates of Burkholderia pseudomallei (Bp), the causative agent of melioidosis, are known to exhibit diverse phenotypic traits, suggesting significant intraspecies genetic heterogeneity. Using whole-genome Bp microarrays, we experimentally mapped patterns of large-scale genomic variation in 93 South East Asian clinical, environmental, and animal Bp isolates. 14% of the reference Bp K96243 genome was variably present across the strain panel, more than double previous estimates, and both hypothetical proteins and paralogous gene pairs (PGPs) were significantly over-represented in the set of strain-variable genes. Examining patterns of PGP retention and loss, we successfully sub-categorized the PGPs into non-redundant, functionally biased, and completely redundant classes. We then identified 20 novel regions (“islands”) variably present between strains previously missed by computational analysis. Three of these novel islands contained lipopolysaccharide (LPS) biosynthesis genes, and strains lacking one such LPS island demonstrated reduced virulence in mouse infection assays. Clinical isolates associated with human melioidosis were strongly associated with the presence of specific genomic islands, but a common set of virulence-related genes was present in all strains. Our results suggest that most Bp strains possess a core virulence machinery capable of causing disease, but accessory functions provided by mobile elements may predispose distinct host species and ecological niches to specific individual strains. This hierarchical model of Bp virulence reconciles previous conflicting studies comparing Bp environmental and clinical isolates, and suggests novel molecular strategies for disease surveillance and outbreak detection efforts in melioidosis. Keywords: aCGH of 93 Bp strains genomic DNA of 93 Bp strains were assessed on Bp_array_v2
Project description:We have found that knockdown of the human telomerase RNA template, hTR, induces Bim-mediated apoptosis independent of telomere length in primary human CD4 T cells, whereas knockdown of the telomerase enzymatic protein, hTERT does not induce apoptosis in the timeframe of our studies. We used microarray analysis to determine any gene changes in human primary CD4 T cells that could provide mechanistic insight into hTR knockdown induced apoptosis. Cells with control shScramble, shTERT, and shTR were used in this experiment Primary human CD4 T cells were trandsuced in biological triplicates with lentivirus containing shRNAs against a scrambled sequence, hTERT, and hTR 24 hours after CD3 CD28 stimiulation. Cells were cultured in the presence of puromycin to select for cells with shRNAs. shTERT and shTR were compared to shScramble results.
Project description:The morphogen Indian Hedgehog plays a very important role during intestinal embryogenesis, but also maintains homeostasis of the adult gut. Intestinal Indian Hedgehog is expressed by the intestinal epithelium and signals in paracrine manner to fibroblasts in the stromal compartment. Unresolved deletion of Ihh from the intestinal epithelium leads to a severe enterocolitis. We studied the short term changes in the colon upon deletion of Ihh from the epithelial layer. Overall design: By using VillinCreERT2-IHH fl/fl mice we specifically deleted Indian Hedgehog from the intestinal epithelium (Villin is expressed by the intestinal epithelial layer). 7 days after the start of induction with tamoxifen colons were isolated.
Project description:Three-dimensional (3D) culture of hepatocytes leads to improved and prolonged synthetic and metabolic functions, but the underlying molecular mechanisms were unknown. In order to investigate the molecular mechanisms underlying 3D cell-cell interactions in maintaining hepatocyte differentiated functions ex vivo, microarray analyses were performed on primary mouse hepatocytes cultured either as monolayers on tissue culture dishes (TCD) or as 3D aggregates in rotating wall vessel (RWV) bioreactors. Primary hepatocytes were isolated from mice and cultured on TCDs or within RWVs for 4 hours or 24 hours. Freshly isolated hepatocytes (t0) were used as baseline controls. Each experimental condition has three independent biological replicates.
Project description:Paneth cells recide in the intestinal crypt bottom and are part of the innate immunity and of the intestinal stem cell niche. We used microarrays to detail the global changes in gene expression following reduced calorie intake. Mice were kept on ad libitum or calorie restricted (60% of calories of ad libitum) diets for 4-7 weeks and paneth cells were isolated using flowcytometry
Project description:DNA methylation is a heritable epigenetic modification involved in gene silencing, imprinting, and the suppression of retrotransposons. Global DNA demethylation occurs in the early embryo and the germline and may be mediated by Tet (ten-eleven-translocation) enzymes, which convert 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC). Tet enzymes have been extensively studied in mouse embryonic stem (ES) cells, which are generally cultured in the absence of Vitamin C, a potential co-factor for Fe(II) 2-oxoglutarate dioxygenase enzymes like Tets. Here we report that addition of Vitamin C to ES cells promotes Tet activity leading to a rapid and global increase in hmC. This is followed by DNA demethylation of numerous gene promoters and up-regulation of demethylated germline genes. Tet1 binding is enriched near the transcription start site (TSS) of genes affected by Vitamin C treatment. Importantly, Vitamin C, but not other antioxidants, enhances the activity of recombinant human Tet1 in a biochemical assay and the Vitamin C-induced changes in hmC and mC are entirely suppressed in Tet1/2 double knockout (Tet DKO) ES cells. Vitamin C has the strongest effects on regions that gain methylation in cultured ES cells compared to blastocysts and in vivo are methylated only after implantation. In contrast, imprinted regions and intracisternal A-particle (IAP) elements, which are resistant to demethylation in the early embryo, are resistant to Vitamin C-induced DNA demethylation. Collectively, this study establishes that Vitamin C is a direct regulator of Tet activity and DNA methylation fidelity in ES cells. Oct4-GiP mouse embryonic stem (ES) cells were cultured in the presence or absence of Vitamin C (L-ascorbic acid 2-phosphate, 200 μg/ml) for 72 hours. RNA was harvested from biological triplicates for each condition and hybridized to Affymetrix microarrays. Cells were maintained in N2B27 medium supplemented with LIF (1000 U/ml), MEK inhibitor PD0325901 (1 μM), and GSK3β inhibitor CHIR99021 (3 μM).
Project description:Identification of genes involved in trophoblast differentiation is of great interest in understanding cellular and molecular mechanisms involved in placental development and is relevant clinically to fetal development, fertility, and maternal health. To understand, on a global scale, changes in the transcriptome during the differentiation of hESCs down the trophoblast lineage, a large-scale microarray analysis was performed. This work provides an in vitro functional genomic model with which to identify genes involved in trophoblast development. We investigated differentiation of human embryonic stemcells (hESCs) down the trophoblast lineage by culture with bone morphogenetic protein 4 (BMP4) over a 10-day period and analyze the cell every 2-days.