Project description:The aim of the experiment was to analyse gene expression profiles in Brca1 tumours arising from different mammary epithelial cell populations use a Cre-loxP based conditional knockout system. K14 promoter driving Cre expression caused Brca1 knockout in basal stem cells and thus stem cell origin tumours whereas Blg promoter driving Cre expression caused Brca1 knockout in luminal progenitor cells and thus progenitor origin tumours. Individual arrays were carried out on labelled cDNA made from RNA isolated from mouse mammary tumours. Only cDNA passing Almac diagnostics QC criteria were hybridised to arrays. Only arrays passing QC criteria after hybrisiation were subsequently analysed.
Project description:Transcriptional analysis of human T cells differentiated in 4 T Helper context ( Th0, Th1, Th2 and Th17) in the presence or not of Interferon alpha We analyzed the transcriptomic profiles of 4 human naives T cells diferentiated in Th0, Th1, Th2 and Th17 in the presence or not of Interferon Alpha. Microarray analyses were performed in 2 time points : 1/ after Day 5 of polarization (= Day5); 2/ after Day 5+ four hours of re-stimulation (=Day 5+ 4H restim) in 3 different donors.
Project description:Microarrays were used to examine gene expression changes that may be present in the fallopian tube epithelium of morphologically normal BRCA1 mutation positive and negative subjects. Fallopian tube epithelia has been implicated as an early point of origin for serous carcninoma. By examining the early events present in the microenvironment of this tissue between BRCA1 mutation carriers and non-carriers, we hoped to elucidate mechanisms that may lead to the development of epithelial ovarian cancer. Laser microcapture of samples from 12 BRCA1 mutation carriers and 12 non-mutation subjects was performed. Samples were further grouped according to menstrual cycle.
Project description:Pericytes are integral components of the tissue vasculature and have essential functions in tumour angiogenesis. Endosialin (CD248) is a type I transmembrane glycoprotein highly expressed on pericytes in the tumour vasculature of most solid tumours, however it is low or negligibly expressed on normal tissue pericytes. Experiments using wild-type and endosialin-knockout mice has revealed that stromal endosialin expression facilitates intravasation of tumor cells from the primary tumor into the circulation, thereby promoting metastatic dissemination. BALB/c Ub-GFP wild-type and endosialin-knockout mice were inoculated with 67NR tumor cells. Tumor pericytes and tumor endothelial cells were isolated from primary tumors by FACSorting and total RNA was extracted from freshly isolated (i.e. without in vitro culture) cell populations.
Project description:Comparison of Mpl-/- mouse LSK cells, either treated with control (GFP) or Mpl lentivirus. Lineage negative bone marrow cells were isolated and transduced and transplanted into Mpl-/- recipient mice. After transplantation and follow up mice were sacrificed and LSK (lineage negative, Sca-1 positive, cKit positive) cells were isolated by FACS. RNA was isolated using RNeasy Micro Kit (Qiagen GmbH, Hilden, Germany) and RNA was amplified for microarray hybridization using the Nugen Ovation system (Nugen Technologies, AC Bemmel, Netherlands). The resulting material was hybridized to Affymetrix Mouse 430 2.0 arrays. RMA normalization and summarization was performed in R 2.10 using Bioconductor packages. The aim was to show the normalization of Mpl associated gene expression. 3 control (GFP transduced) samples of Mpl -/- mouse LSK cells and 3 treatment (Mpl transduced) samples of Mpl -/- mouse LSK cells.
Project description:Gene expression profiling of 273 pre-treatment endoscopic FFPE oesophageal and oesophago-gastric junctional adenocarcinomas for validation of a DNA Damage Response Deficiency Assay. All patients were treated with neo-adjuvant chemotherapy followed by surgical resection and were collected at four UK centres in the Oesophageal Cancer Clinical and Molecular Stratification (OCCAMS) consortium between 2004 and 2013. Biopsies were reviewed for pathological subtype prior to marking for macrodissection and samples containing at least 50% adenocarcinoma tissue by area were taken forward. Total RNA was extracted using the RecoverallTM Total Nucleic Acid Isolation Kit for FFPE (Thermo Fisher Scientific, Waltham, MA) and amplified using the NuGen Ovation FFPE Amplification System v3 (NuGen San Carlos, CA). The amplified product was hybridized to the Almac Diagnostics Xcel™ array (Almac, Craigavon, United Kingdom), a cDNA microarray-based technology optimized for archival FFPE tissue, and analysed using the Affymetrix 7G scanner (Affymetrix, Santa Clara, CA)
Project description:Despite the high prevalence and poor outcome of patients with metastatic lung cancer, the mechanisms of tumour progression and metastasis remain largely uncharacterized. We modelled human lung adenocarcinoma, which frequently harbours activating point mutations in KRAS1 and inactivation of the p53-pathway2, using conditional alleles in mice3-5. Lentiviral-mediated somatic activation of oncogenic Kras and deletion of p53 in the lung epithelial cells of KrasLSL-G12D/+;p53flox/flox mice initiates lung adenocarcinoma development4. Although tumours are initiated synchronously by defined genetic alterations, only a subset become malignant, suggesting that disease progression requires additional alterations. Identification of the lentiviral integration sites allowed us to distinguish metastatic from non-metastatic tumours and determine the gene expression alterations that distinguish these tumour types. Cross-species analysis identified the NK-2 related homeobox transcription factor Nkx2-1 (Ttf-1/Titf1) as a candidate suppressor of malignant progression. In this mouse model, Nkx2-1-negativity is pathognomonic of high-grade poorly differentiated tumours. Gain- and loss-of-function experiments in cells derived from metastatic and non-metastatic tumours demonstrated that Nkx2-1 controls tumour differentiation and limits metastatic potential in vivo. Interrogation of Nkx2-1 regulated genes, analysis of tumours at defined developmental stages, and functional complementation experiments indicate that Nkx2-1 constrains tumours in part by repressing the embryonically-restricted chromatin regulator Hmga2. While focal amplification of NKX2-1 in a fraction of human lung adenocarcinomas has focused attention on its oncogenic function6-9, our data specifically link Nkx2-1 downregulation to loss of differentiation, enhanced tumour seeding ability, and increased metastatic proclivity. Thus, the oncogenic and suppressive functions of Nkx2-1 in the same tumour type substantiate its role as a dual function lineage factor. 23 cell lines derived from primary tumor or metastasis. 6 samples analyzed to determine the effect of Nkx2-1 knockdown on gene expression
Project description:Thrombopoietin (Thpo) signals via its receptor Mpl and regulates megakaryopoiesis, hematopoietic stem cell (HSC) maintenance and post-transplant expansion. Mpl expression is tightly controlled and deregulation of Thpo/Mpl-signaling is linked to hematological disorders. Here, we constructed an intracellular-truncated, signaling deficient Mpl protein which is presented on the cell surface (dnMpl). The transplantation of bone marrow cells retrovirally transduced to express dnMpl into wildtype mice induced thrombocytopenia, and a progressive loss of HSC. Functional analysis of the truncated Mpl in vitro and in vivo demonstrated no internalization after Thpo binding and the inhibition of Thpo/Mpl-signaling in wildtype cells due to dominant-negative (dn) effects by receptor competition with wildtype Mpl for Thpo binding. Gene expression analysis was performerd of Lin-Sca1+cKit+ (LSK) cells isolated from mice transplanted with dnMpl transduced BM cells. The gene expression profile supported the exhaustion of HSC due to increased cell cycle progression and identified new and known downstream effectors of Thpo/Mpl-signaling in HSC (namely TIE2, ESAM1 and EPCR detected on the HSC-enriched LSK cell population). In summary, we established a novel way of Thpo/Mpl inhibition in the adult mouse and performed in depth analysis of the phenotype including gene expression profiling. BM cells were flushed from the femurs and tibias of C57Bl/6 donor mice. Lineage-marker negative (lin-) cells were isolated by magnetic cell sorting using lineage-specific antibodies (GR1, CD11b, CD45R/B220, CD3e, TER-119). Prior to viral transduction, lin- BM cells were prestimulated for 24-48h in StemSpan, containing 10 ng/ml murine SCF, 20 ng/ml murine Thpo, 10 ng/ml recombinant human FGF-1, 20 ng/ml murine IGF2, 1% penicillin/streptomycin, 2 mM glutamine. Lin- cells were transduced twice on two following days with an MOI of 10 with retroviral vectors on Retronectin coated and preloaded wells (10 μg/cm2). On day four after isolation, 5x10^5 cells/mouse were intravenously injected into lethally irradiated C57Bl/6 mice (10 Gy). Eight weeks after transplantation Lin-Sca1+cKit+ (LSK) cells were isolated from dnMpl and control transplanted mice by fluorescent activated cell sorting. For each sample, BM of 2-5 mice were pooled. RNA was isolated using RNeasy Micro Kit (Qiagen GmbH, Hilden, Germany). RNA quality was assessed using the Agilent 2100 Bioanalyzer.
Project description:The transcription factor Evi1 is essential for the formation and maintenance of hematopoietic stem cells, and induces clonal dominance with malignant progression upon constitutive activation by chromosomal rearrangements or transgene integration events. To understand the immediate and adaptive response of primary murine hematopoietic cells to the transcriptional upregulation of Evi1, we developed an inducible lentiviral vector system with a robust expression switch. We found that Evi1 delays differentiation and promotes survival in myeloid culture conditions, orchestrating a battery of genes involved in stemness (Aldh1a1, Ly6a [Sca1], Abca1, Epcam, among others). Importantly, Evi1 suppresses Cyclins and Cyclin-dependent kinases (Cdk), while it upregulates Cdk inhibitors, inducing quiescence in various proliferation-inducing cytokine conditions and operating in a strictly dose-dependent manner. Hematopoietic cells with persisting Evi1-induction tend to adopt a relatively low expression level. We thus classify Evi1 as a dormancy-inducing oncogene, likely requiring epigenetic and genetic compensation for cell expansion and malignant progression. Evi1 dependent myeloid clone C6 cells expressing a Doxycyclin inducible codon-optimized murine Evi1 were harvested at day 2 after DOX withdrawal and compared to C6 cells under Doxycyclin 1 ug/ml
Project description:We propose a new pipeline for the refinement of spectrum-peptide assignment, designed specifically for MHC ligand identification. By modeling the peptidome as a collection of a limited number of specificities, corresponding to the MHC alleles of the cell line, our method achieves increased sequencing depth, while at the same time removing potential experimental outliers and contaminants.