ABSTRACT: Many preclinical therapy studies have focused on a small number of well-described mouse allograft or human xenograft models that poorly represent the heterogeneity of human disease. Here we have assembled a panel of mouse mammary cell lines that metastasize in syngeneic mouse hosts and we have assessed gene expression programs in the untreated primary tumors with the goal of generating information that may be useful to the identification of biomarkers that predict response to therapeutic intervention. We used microarrays to assess global gene expression programs in primary tumors from 12 metastatic mouse mammary tumor models transplanted orthotopically into syngeneic, fully immunocompetent mouse hosts. The 12 tumor models used here are based on published cell lines that had been established from either spontaneous mammary tumors or from mammary tumors arising in genetically engineered mouse models. All cell lines were previously described to be metastatic. Cells were surgically implanted in the #4 mammary fat pads of syngeneic mice and primary tumors were harvested when they reached 0.5-1.0 cm diameter and snap-frozen for later RNA extraction. 4 independent tumors were collected for each of the 12 models.
Project description:Anaplastic thyroid carcinoma (ATC) has among the worst prognosis of any solid malignancy. The low incidence of the disease has in part precluded systematic clinical trials and tissue collection, and there has been little progress in developing effective therapies. BRAF and TP53 mutations co-occur in a high proportion of ATC, particularly those associated with a precursor papillary thyroid carcinoma (PTC). In order to develop an adult-onset model of BRAF-mutant anaplastic thyroid carcinoma, we generated a novel thyroid-specific CreER transgenic mouse. We utilize a Cre-regulated BrafV600E mouse and a conditional Trp53 allelic series to demonstrate that p53 constrains progression from papillary to anaplastic thyroid carcinoma. Gene expression and immunohistochemical analyses of murine tumors identified the cardinal features of human ATC including loss of differentiation, local invasion, distant metastasis and rapid lethality. We employed small animal ultrasound imaging to monitor autochthonous tumors, and show that treatment with the selective BRAF inhibitor PLX4720 improved survival, but did not lead to tumor regression or suppress signaling through the MAPK pathway. Combination of PLX4720 and the MEK inhibitor PD0325901 more completely suppressed MAPK pathway activation in mouse and human ATC cell lines, and improved the structural response and survival of ATC-bearing animals. This model expands the limited repertoire of autochthonous models of clinically aggressive thyroid cancer, and these data suggest that small molecule MAPK pathway inhibitors hold clinical promise in the treatment of advanced thyroid carcinoma. Total RNA from five murine papillary thyroid carcinoma (PTC) tumors and five murine anaplastic thyroid carcinoma (ATC) tumors was analyzed.
Project description:The transcriptome of naive OT-I T cells was compared to memory CD8 T cells after 1, 2, 3, or 4 infection with ovalbumin expressing Listeria monocytogenes (LM-OVA). Naive Thy1.1 OT-I T cells were adoptively transferred into Thy1.2 naive hosts prior to infection with LM-OVA. The resulting memory CD8 T cell population was again adoptively transferred into naive hosts and the recipient mice were again infected with LM-OVA. The adoptive transfer was repeated up to four times to generate memory CD8 T cells with up to four consecutive antigen stimulations. Three individual mice were analyzed for each group. For quaternary memory CD8 T cells, spleens from two to three mice were pooled for each sample. Naive OT-I T cells served as control samples. http://dx.doi.org/10.1016/j.immuni.2010.06.014
Project description:Embryonic development is tightly regulated by transcription factors and chromatin-associated proteins. H3K4me3 is associated with active transcription and H3K27me3 with gene repression, while the combination of both keeps genes required for development in a plastic state. Here we show that deletion of the H3K4me2/3 histone demethylase Jarid1b (Kdm5b/Plu1) results in major neonatal lethality due to respiratory failure. Jarid1b knockout embryos have several neural defects including disorganized cranial nerves, defects in eye development and increased incidences of exencephaly. Moreover, in line with an overlap of Jarid1b and Polycomb targets genes, Jarid1b knockout embryos display homeotic skeletal transformations typical for Polycomb mutants. Genome-wide analysis demonstrated that normally inactive genes encoding developmental regulators acquire aberrant H3K4me3 in early Jarid1b knockout embryos. H3K4me3 accumulates as embryonic development proceeds, leading to increased expression of neural master regulators in knockouts. Taken together, these results suggest that Jarid1b contributes to mouse development by protecting developmental genes from inappropriate acquisition of active histone modifications. * Lack of Jarid1b leads to major neonatal lethality and defects in neural systems * Jarid1b mutants display homeotic skeletal transformations * H3K4me3 is increased at inactive transcriptional regulators in Jarid1b-/- embryos * Chromatin changes are accompanied by elevated levels of key neural transcription factors RNA was extracted from heads of E8.5 embryos, three pools of 4 heads each were used per genotype
Project description:Liver dysfunction and cirrhosis affect vasculature in several organ systems and cause impairment of organ functions, thereby increasing morbidity and mortality. If a mouse model of hepatopulmonary syndrome (HPS) could be established, greater insight into the genetic basis of the disease would be gained. Our objectives were to establish a mouse model of lung injury after common bile duct ligation (CBDL) and to investigate pulmonary pathogenesis for application in future therapeutic approaches. Balb/c mice were subjected to CBDL. Immunohistochemical analyses and real-time quantitative reverse transcriptional polymerase chain reaction were performed on pulmonary tissues. The presence of HPS markers were detected by western blot and microarray analyses. We observed extensive proliferation of CD31-positive pulmonary vascular endothelial cells 2 weeks after CBDL, and identified 11 up-regulated and 8 down-regulated proteins that were associated with angiogenesis. MMP-9 protein was highly expressed at 3 weeks after CBDL, and less expressed in lungs of the control group. Contrary to our expectation, lung pathology in our mouse model exhibited differences from that of rat models, and the mechanisms responsible for these differences are unknown. This phenomenon may be explained by contrasting processes related to TNF induction of angiogenic signaling pathways in the inflammatory phase; thus, we suggest that our mouse model can be applied to pulmonary pathological analyses in the inflammatory phase, i.e., to systemic inflammatory response syndrome, acute lung injury, and MOD syndrome. After induction of anesthesia, a median abdominal incision was made and the common bile duct was identified. The duct was dissected carefully under a microscope, and doubly ligated with 7-0 Prolene and transected. In the sham operation (control) group, the duct was dissected without common bile duct ligation. Mice were sacrificed at 2 and 3 weeks after surgery. CD31-positive cells were assembled from three mice in each group.
Project description:p53-repressed transcripts have recently been shown to play important roles in various biological processes, such as stem cell differentiation and cancer. We identified a transcript named Apela that is repressed by p53 and highly expressed in mouse ES cells. To see which transcripts are affected by Apela knockdown, we performed gene expression microarray using Affymetrix Gene ST 1.0 array. Two short hairpin RNAs (shRNAs) targeting Apela were used to decrease the RNA levels of Apela to about 20% of the control (a shRNA target luciferase, shLuc). We designed two lentivirus-based shRNAs against Apela and used shRNA against luciferase as a control. Lentiviruses were made and used to transduced mouse ES cells. For each shRNA, three repeats were done.
Project description:This SuperSeries is composed of the following subset Series: GSE30429: Gene Array Analyzer (GAA): Alternative usage of gene arrays to study alternative splicing events (MoGene array) GSE32998: Gene Array Analyzer (GAA): Alternative usage of gene arrays to study alternative splicing events (MoEx array) Refer to individual Series
Project description:The latest version of microarrays released by Affymetrix, the GeneChip Gene 1.0 ST Arrays (gene arrays), are designed in a similar fashion as exon arrays, which enables to identify differentially expressed exons, rather than only the expression level of whole transcripts. Here, we propose an extension, Gene Array Analyzer (GAA), to our previously published Exon Array Analyzer (EAA). GAA enables to analyse gene arrays on exon level and therefore supports to identify alternative splicing with gene arrays. To show the applicability of GAA, we used gene arrays to profile alternative splice events during the development of the heart. Further re-analysis of published gene arrays could show, that some of these splice events reoccur under pathological conditions. The web interface of GAA is user friendly, functional without set up and freely available at http://GAA.mpi-bn.mpg.de. Alternative splicing and gene expression analysis during development of the heart and cardiomyoyte differentiation.
Project description:Ten-eleven translocation (Tet) family of DNA dioxygenases converts 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5- carboxylcytosine (5caC) through iterative oxidation reactions. While 5mC and 5hmC are relatively abundant, 5fC and 5caC are at very low levels in the mammalian genome. Thymine DNA glycosylase (TDG) and base excision repair (BER) pathways can actively remove 5fC/5caC to regenerate unmethylated cytosine, but it is unclear to what extent and at which part of the genome such active demethylation processes take place. Here, we have performed high-throughput sequencing analysis of 5mC/5hmC/5fC/5caC- enriched DNA using modification-specific antibodies and generated genome-wide distribution maps of these cytosine modifications in wild-type and Tdg-deficient mouse embryonic stem cells (ESCs). We observe that the steady state 5fC and 5caC are preferentially detected at repetitive sequences in wild-type mouse ESCs. Depletion of TDG causes marked accumulation of 5fC and 5caC at a large number of distal gene regulatory elements and transcriptionally repressed/poised gene promoters, suggesting that Tet/TDG-dependent dynamic cycling of 5mC oxidation states may be involved in regulating the function of these regions. Thus, comprehensive mapping of 5mC oxidation and BER pathway activity in the mammalian genome provides a promising approach for better understanding of biological roles of DNA methylation and demethylation dynamics in development and diseases. Gene expression comparison of control and Tdg knockdown mouse embryonic stem cells.
Project description:Ten-eleven translocation (Tet) family of DNA dioxygenases converts 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5- carboxylcytosine (5caC) through iterative oxidation reactions. While 5mC and 5hmC are relatively abundant, 5fC and 5caC are at very low levels in the mammalian genome. Thymine DNA glycosylase (TDG) and base excision repair (BER) pathways can actively remove 5fC/5caC to regenerate unmethylated cytosine, but it is unclear to what extent and at which part of the genome such active demethylation processes take place. Here, we have performed high-throughput sequencing analysis of 5mC/5hmC/5fC/5caC- enriched DNA using modification-specific antibodies and generated genome-wide distribution maps of these cytosine modifications in wild-type and Tdg-deficient mouse embryonic stem cells (ESCs). We observe that the steady state 5fC and 5caC are preferentially detected at repetitive sequences in wild-type mouse ESCs. Depletion of TDG causes marked accumulation of 5fC and 5caC at a large number of distal gene regulatory elements and transcriptionally repressed/poised gene promoters, suggesting that Tet/TDG-dependent dynamic cycling of 5mC oxidation states may be involved in regulating the function of these regions. Thus, comprehensive mapping of 5mC oxidation and BER pathway activity in the mammalian genome provides a promising approach for better understanding of biological roles of DNA methylation and demethylation dynamics in development and diseases. Refer to individual Series
Project description:Leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1) is a pan-ErbB negative regulator and intestinal stem cell marker downregulated in many malignancies. Over 91% Lrig1-CreERT2/CreERT2 (Lrig1-/-) mice developed duodenal adenomas, providing the first in vivo evidence Lrig1 acts as a tumor suppressor. We thus characterize the differential expressing transcripts in these duodenal adenomas to explore the pathegenesis. Elevated expression of the Egfr ligands was detected in adenomas compared to adjacent normal tissue. These adenomas also expressed the gastric-specific genes Gastrokine1 and Mucin5ac, indicating gastric metaplasia. In this dataset, we include the expression data obtained from proximal adenomas as well as adjacent normal tissue and wildtype proximal duodenum. These data are used to obtain 679 upregulated and 874 downregulated transcripts in tumors. Total RNA from matched duodenal tumors or adjacent normal or wildtype duodenum was isolated with Rneasy (with Dnase digestion) and used to probe the Affymetrix Mouse Gene 1.0 ST chip; the probe was prepared with Ambion Expression Target preparation Kit P/N 4411974.