Study of gene expression of mouse cMET experiments
ABSTRACT: We analyzed the molecular changes in a mouse c-MET over-expression model of hepatocellular carcinoma (HCC). Overall design: FVB mice overexpressing human c-MET carried one copy of the LAP-tTa transgene (the liver-specific LAP promoter driving the Tet-VP16 transactivator) and one copy of the TRE-c-MET transgene (Tet-operator regulated human c-MET gene). Normal liver or liver tumor tissue (two per mouse) was collected and processed for gene expression profiling.
Project description:We analyzed the molecular changes in a mouse c-MET over-expression model of hepatocellular carcinoma (HCC). FVB mice overexpressing human c-MET carried one copy of the LAP-tTa transgene (the liver-specific LAP promoter driving the Tet-VP16 transactivator) and one copy of the TRE-c-MET transgene (Tet-operator regulated human c-MET gene). Normal liver or liver tumor tissue (two per mouse) was collected and processed for gene expression profiling.
Project description:Gene expression changes were examined in transgenic MYC-driven liver cancers at different time points as tumors formed and upon early regression. Time points evaluated include: Control (non-tumor bearing), Pre-tumor (mice were removed from doxyclycine in their diet to induce MYC oncogene expression for 4-5 weeks), Tumor (tumor nodules from mice that had been off of doxycycline for 8-9 weeks) and Early tumor regression (tumor-bearing mice were placed back on doxycycline for 72 hrs to inhibit MYC oncogene expresion). MYC-Driven Mouse Tumor Models are described in Schahaf, et al., Nature, 2004 and Goga, et al., Nature Medicine, 2007. Overall design: 11 Total Samples: 3 Control, 4 Pre-Tumor, 4 Tumor, 4 Early Tumor Regression. Control = LAP-tTA transgenic mice. Others = TRE-MYC x LAP-tTA double transgenic mice (doxy off).
Project description:Gene expression changes were examined in transgenic MYC-driven liver cancers at different time points as tumors formed and upon early regression. Time points evaluated include: Control (non-tumor bearing), Pre-tumor (mice were removed from doxyclycine in their diet to induce MYC oncogene expression for 4-5 weeks), Tumor (tumor nodules from mice that had been off of doxycycline for 8-9 weeks) and Early tumor regression (tumor-bearing mice were placed back on doxycycline for 72 hrs to inhibit MYC oncogene expresion). MYC-Driven Mouse Tumor Models are described in Schahaf, et al., Nature, 2004 and Goga, et al., Nature Medicine, 2007. 11 Total Samples: 3 Control, 4 Pre-Tumor, 4 Tumor, 4 Early Tumor Regression. Control = LAP-tTA transgenic mice. Others = TRE-MYC x LAP-tTA double transgenic mice (doxy off).
Project description:c-Fos, a member of the stress-activated Activator Protein 1 (AP-1) transcription factor family, is expressed in human hepatocellular cancer (HCC). Using genetically engineered mouse models (GEMMs) we show that hepatocyte-specific expression of c-Fos leads to a proliferative, de-differentiated phenotype, whereas hepatocyte-specific deletion of c-Fos protects against diethylnitrosamine (DEN)-induced liver cancer. Furthermore, c-Fos-expressing livers resemble human HCCs based on expression profiles. In the present RNA seq, we intend to analyze the transcriptomic profile of livers at 2 and 4 mo hepatocyte-specific c-Fos expression compared to the corresponding age-matched control mice. Moreover, we analyzed livers of mice with hepatocyte-specific deletion c-Fos at 48h after DEN treatment compared to identically treated control mice. Overall design: The general idea was to analyze the transcriptomic profile of hepatocyte-specific c-Fos over-expressing livers at 2 and 4 mo expression. Hereby, a hepatocyte-specific doxycycline (Dox)-switchable mouse model was (LAP-tTA; col1a1:Tet-O-fosFlag) was generated and c-Fos expression was induced at the age of 3 weeks by removal of doxycycline. Each sample LaptTA-fos-MUT represents an individual hepatocyte-specific c-fos expressing mouse at the indicated time-point and the corresponding identically treated control mouse LaptTA-fos-CO. Moreover, the transcriptomic profile of livers with hepatocyte-specific deletion of c-Fos at 48h after diethylnitrosamine (DEN)-induced liver cancer initiation was analyzed. For hepatocyte-specific knock-out of c-Fos, mice with conditional alleles of c-fos and the Alfp-Cre transgene were used. Control mice only carried the Alfp-Cre transgene. At the age of 8 weeks these mice were injected with 100mg/kg DEN. Each sample AlfpCre-fos-MUT_DEN represents an individual hepatocyte-specific c-fos knock-out mouse 48h after DEN and the identically treated control mouse AlfpCre-fos-CO-Cre+_DEN.
Project description:Mutation in TDP-43 is causative to amyotrophic lateral sclerosis (ALS). TDP-43 is a multifunctional ribonucleoprotein and is reproted to regulate thousands of genes in neurons, but how astrocytes contribute to TDP-43 pathogenesis is not known. This study examined how mutant TDP-43 in astrocytes kills motor neurons and causes ALS phenotypes. Primary astrocytes were isolated from transgenic rats expressing mutant TDP-43 or from control rats without mutant TDP-43 expression. Cultured astrocytes were induced to express mutant human TDP-43 and their gene expression profiles were determined by microarray assays. Microarray analysis revealed that hundreds of genes were altered in astrocytes in response to mutant TDP-43 expression. As mutant TDP-43 transgene is under the control of tetracycline-regulated pomoter elements (TRE), mutant TDP-43 expression is subjected to Doxycline regulation. Astrocytes isolated from GFAP-tTA/TRE-TDP43M337V rats were desiginated as M337V groups and astrocytes isolated from GFAP-tTA single transgenic rats were desiginated as tTA control groups. Total RNA was isolated from cultured astrocytes at varying times (3, 4, or 6 days after Dox withdrawal) after mutant TDP-43 was induced in astrocytes. Upon mutant TDP-43 induction in astroyctes, gene expression profiles in astroyctes were determined by Illumina Direct Hybridization Assay and compared between tTA and M337V groups at the varying time points of mutant TDP-43 induction.
Project description:Chromosomal translocations encoding the MLL-AF9 and MLL-ENL fusion transcription factors are prevalent in infant acute leukaemia and therapy-related leukaemia. In order to conditionally express the MLL-fusion oncogene in primary haematopoietic progenitor cells (HPC), retroviral delivery of the Tet-off expression system was used (Horton et al., Cancer Res, 2005). Treatment of the conditional cells with Doxycycline caused a decrease in MLL-AF9/ENL mRNA and protein expression, and resulted in terminal differentiation of the cells. By analysing global changes in gene expression after treatment of cells with Doxycycline we were able to identify a number of potential transcriptional target genes of the MLL-AF9 and MLL-ENL fusion oncogenes. Overall design: Lineage negative progenitors were purified from murine bone marrow and co-transduced with MSCV-TRE-MLL-AF9 or MSCV-TRE-MLL-ENL and MSCV-tTA retroviral supernatants. Six independent cell lines (MA1, MA3, MA4, ME4, ME5, ME7) with conditional expression of the MLL-AF9 or MLL-ENL oncogene and two independent cell lines (cMA3, cME3) with constitutive MLL-fusion oncogene expression were generated. The immortalised cell lines were characterised to determine their tTA dependent MLL-fusion oncogene expression, morphology, immunophenotype, and cytokine requirements. Total RNA was extracted, using TRIzol reagent, from the cell lines, each of them cultured without or with 2µg/ml Doxycycline for 48 hours and used for hybridisation on Affymetrix microarrays.
Project description:Our goal was to examine whether the HIV Tat peptide, which is usually secreted from infected cells and has the potential to act in other cell types, alters gene expression in the Central Nervous System, and whether a drug abuse co-morbidity, in the case Methamphetamine, can play a role in further modifying gene expression. In order to address the effects of HIV Tat and Methamphetamine, alone and combined, we used an in vivo mouse model that has been described to mimic several aspects of neuroHIV, including changes in inflammatory markers, and decreased expression of dopamine receptors. These animals are transgenic mice, which upon treatment with with doxycycline for 10 days, express TAT protein under the control of the glial fibrilary associated protein (GFAP) promoter in the brain. They were treated with Meth and Saline for identification of gene expression changes that result from Tat or Methamphetamine alone, or from their interaction. There was an overall suppression of gene expression by Methamphetamine, in Tat- mice. The expression of Tat caused most Meth-induced changes to remain at control levels. Overall design: A total of 20 male mice, 6 weeks old, 10 containing the GFAP promoter-controlled Tet-binding protein (TAT−) and 10 containing both the GFAP promoter-controlled Tet-binding protein and the TRE promoter-TAT protein transgene (TAT+) were tested. Inducible Tat transgenic mouse colonies with a C57BL/6J background are obtained by generation of two separate transgenic lines Teton-GFAP mice and TRE-Tat86 mice, and then cross-breeding of these two transgenic mouse lines, as previously described (Kim et al., 2003). The mice were housed in groups of 2–4 in a humidity- and temperature-controlled animal facility on a 12 h/12 h reverse light/dark cycle (lights off at 7:00 AM) with ad libitum access to food and water. All mice were treated with a doxycycline regimen (doxycycline hyclate; Sigma) of 100 mg/kg, intraperitoneally, once a day for 7 days. This regimen is based on the previously demonstrated efficacy of Tat induction at this dose of doxycycline (Carey et al., 2012; Paris et al., 2014a). Only mice containing both the GFAP promotor-controlled Tet-binding protein and the TRE promoter-TAT protein transgene (TAT+) generate TAT protein after doxycycline administration. Control TAT- animals had the GFAP promoter-controlled Tet-binding protein, but did not have TRE promoter-TAT protein transgene. All mice (TAT+ and TAT-) were administered doxycycline injections in the evening (17:00 h), beginning the day before the first injection of Methamphetamine or Saline. The Methamphetamine administration procedure followed a sensitization paradigm, and consisted of seven consecutive days of intraperitoneal injection with either saline (0.9%) or 2 mg/kg methamphetamine (methamphetamine hydrochloride; Sigma, St. Louis, MO, USA). The animals were anesthesized and perfused with ice cold PBS 24 hrs after the last injection, and the brains were snap frozen for the determination of changes in the gene expression. The animals were divided in 4 groups: G1=Tat-Meth+, G2=Tat-Meth-, G3=Tat+Meth+, G4=Tat+Meth-, and 5 mice per group.
Project description:Analysis of RNA polymerase II (RNAP II) binding data in HCT116-C52-tTA cells provides insight into the stalling site of RNAP II in the human PTPRN2 gene. Overall design: We established a stable HCT116 cell line, HCT116-C52, in which a tetracycline (tet)-repressible promoter was integrated into the second intron of the PTPRN2 gene by homologous recombination. An EGFP-tagged tTA (EGFP-tTA) expression cassette was then introduced into the HCT116-C52 cells by virus transduction to generate HCT116-C52-tTA cells. RNAP II binding sites in HCT116-C52-tTA cells were determined by RNAP II ChIP-seq, using an antibody targeting the YSPTSPS repeats in the C-terminal domain (CTD) of the largest subunit of RNAP II.
Project description:Experiments used RNAseq data to compare transcriptomes of primary cells from different genetic backgrounds cultured under EpiCult and CRC conditions to each other and to allografts grown from these primary cells in nude mice Overall design: Paired-end mRNA-seq with WT, Esr1 over-expressing (CERM, tetracycline-operator(tet-op)-Esr1MMTV-rtTA), CYP19A1 over-expressing (AROM, tet-op-CYP19A1MMTV-rtTA) and Brca1 KO (BRCA, Brca1fl11/fl11/MMTV-Cre/p53+/- ) mice
Project description:Liver transplantation is the only therapeutic option for patients with end-stage liver disease. The shortage of donor organs has led to the search for alternative therapies to restore liver function and bridge patients to transplantation. Our previous work has shown that the proliferation of late gestation E19 fetal hepatocytes is mitogen-independent. This is manifested as differences in the control of ribosome biogenesis, global translation, cell cycle progression and gene expression. In the present study, we investigated whether E19 fetal hepatocytes would engraft and repopulate an injured adult liver. Methods: Fetal hepatocytes were isolated using a monoclonal antibody against a hepatic surface protein, leucine amino peptidase (LAP). LAP+ and LAP- fractions were analyzed by immunofluorescence and microarray. Immunopurified E19 liver cells from DPPIV+ F344 rats were transplanted via splenic injection into partial hepatectomized DPPIV- rats that had been pretreated with mitomycin C. Results: Phenotypic characterization of the LAP+ fetal hepatocytes revealed that more than a third of the isolated cells expressed ductal markers. Transcriptomic analysis revealed that these dual expressing cells represent a distinct subpopulation of less well differentiated hepatocytes. Transplanted immunopurified LAP+ late gestation fetal hepatocytes formed small hepatic, endothelial and occasional ductal colonies within one month. The average size of the colonies derived from the LAP+ cells increased so that by 10 months up to 35% of the liver was repopulated by donor-derived cells. Conclusions: Our studies show that late gestation fetal hepatocytes, despite their being far along in the differentiation process, possess the capacity for extensive liver repopulation. This is likely related to the unexpected presence of a significant proportion of hepatocyte marker-positive cells maintaining a less well differentiated phenotype. Overall design: Two subpopulations of E19 fetal rat hepatocytes were isolated using monoclonal antibodies against the hepatic cell surface marker leucine aminopeptidase and the ducal marker OC.2. Adult hepatocytes were also isolated. RNA was isolated from triplicate biological replicates of fetal LAP+/OC.2- and LAP+/OC.2+ cells as well as adult hepatocytes using the mirvana kit. Affymetrix Rat ST 1.0 arrays were utilized.