Project description:A high percentage of potential oncology drugs fail in clinical trials, partly because preclinical models used to test them are inadequate. Breast cancer is the leading cause of cancer-related death among women worldwide but we lack appropriate in vivo models for the ER+ subtypes, which represent more than 75% of all cases. We address these issues by xenografting tumor cells to their site of origin, the milk ducts. All ER+ cell lines and patient-derived xenografts grow mimicking their clinical counterparts. Disease progresses with invasion and metastasis, which become amenable to study. The action of hormones, important in breast carcinogenesis, can now be studied in a relevant context. Importantly, these open opportunities for development and evaluation of therapies. Eight- to twelve-week-old female SCID Beige mice (Charles River) were injected with 5x10e5 BT20-GFP/luc2 cells (n=3) or 5x10e5 HCC1806-GFP/luc2 cells (n=3) either into the mammary fat pad or 2x10e5 BT20-GFP/luc2 cells (n=3) or 2x10e5 HCC1806-GFP/luc2 cells intraductally (n=3). Xenografted BT20 and HCC1806 basal breast cancer cells were sorted by FACS based on GFP expression; total RNA was extracted using Trizol Reagent (Invitrogen), purified with the miRNeasy Mini Kit (Qiagen), quantity and quality were assessed by NanoDrop®ND-1000 spectrophotometer and RNA 6000 NanoChips with the Agilent 2100 Bioanalyzer (Agilent, Palo Alto, USA). Only samples with RIN score >7.0 were included. For each sample, 300 ng of total RNA were amplified using the message amp II enhanced kit (AM1791, Ambion). 12.5 μg of biotin-labelled cRNA were chemically fragmented. Affymetrix GeneChip Human Genome U133A 2.0 Arrays (Affymetrix, Santa Clara, CA, USA) were hybridized with 11μg of fragmented target, at 45°C for 17 hours, washed and stained according to Affymetrix GeneChip® Expression Analysis Manual (Fluidics protocol FS450_0007). Arrays were scanned using the GeneChip® Scanner 3000 7G (Affymetrix) and raw data was extracted from the scanned images and analyzed with the Affymetrix Power Tools software package (Affymetrix). All statistical analyses were performed using R and Bioconductor packages (http://www.Bioconductor.org). Hybridization quality was assessed using the Expression Console software (Affymetrix). Normalized expression signals were calculated from Affymetrix CEL files using RMA. Differential hybridized features were identified using Bioconductor package â??limmaâ?? that implements linear models for microarray data (Smyth, 2004). P values were adjusted for multiple testing with Benjamini and Hochbergâ??s method to control false discovery rate (FDR) (Benjamini et al., 2001). Probe sets showing â?¥2-fold change and a FDR â?¤0.05 were considered significant.
Project description:A high percentage of potential oncology drugs fail in clinical trials, partly because preclinical models used to test them are inadequate. Breast cancer is the leading cause of cancer-related death among women worldwide but we lack appropriate in vivo models for the ER+ subtypes, which represent more than 75% of all cases. We address these issues by xenografting tumor cells to their site of origin, the milk ducts. All ER+ cell lines and patient-derived xenografts grow mimicking their clinical counterparts. Disease progresses with invasion and metastasis, which become amenable to study. The action of hormones, important in breast carcinogenesis, can now be studied in a relevant context. Importantly, these open opportunities for development and evaluation of therapies. Eight- to twelve-week-old female SCID Beige mice (Charles River) were injected with 2x10e5 MCF7-DsRed/luc2 cells (n=3) either into the mammary fat pad or 5x10e4 MCF7-DsRed/luc2 cells intraductally (n=3). Xenografted MCF7 were FACS sorting based on DsRed expression; total RNA was extracted using Trizol Reagent (Invitrogen), purified with the miRNeasy Mini Kit (Qiagen), quantity and quality were assessed by NanoDrop®ND-1000 spectrophotometer and RNA 6000 NanoChips with the Agilent 2100 Bioanalyzer (Agilent, Palo Alto, USA). Only samples with RIN score >7.0 were included. For each sample, 300 ng of total RNA were amplified using the message amp II enhanced kit (AM1791, Ambion). 12.5 μg of biotin-labelled cRNA were chemically fragmented. Affymetrix GeneChip Human Genome U133A 2.0 Arrays (Affymetrix, Santa Clara, CA, USA) were hybridized with 11μg of fragmented target, at 45°C for 17 hours, washed and stained according to Affymetrix GeneChip® Expression Analysis Manual (Fluidics protocol FS450_0007). Arrays were scanned using the GeneChip Scanner 3000 7G (Affymetrix) and raw data was extracted from the scanned images and analyzed with the Affymetrix Power Tools software package (Affymetrix). All statistical analyses were performed using R and Bioconductor packages (http://www.Bioconductor.org). Hybridization quality was assessed using the Expression Console software (Affymetrix). Normalized expression signals were calculated from Affymetrix CEL files using RMA. Differential hybridized features were identified using Bioconductor package âlimmaâ that implements linear models for microarray data (Smyth, 2004). P values were adjusted for multiple testing with Benjamini and Hochbergâs method to control false discovery rate (FDR) (Benjamini et al., 2001). Probe sets showing â¥2-fold change and a FDR â¤0.05 were considered significant.
Project description:Liver clock regulates transcription of hepatic genes in response to feeding. To explore the possibility that the microbiome influences this process, we measured the liver transcriptome in normal mice (Specific Pathogen Free or SPF mice) and compared it to the transcriptome in mice lacking microbiota (Germ Free or GF mice) at different time points over 24h. We used microarrays to detail the global programme of gene expression in liver of GF and SPF 10-12 weeks-old male C57Bl/6 male mice. There are 40 liver samples, each from an individual mouse. The samples are from germ free mice (GF) and specific pathogen free mice (SPF). Mice of both types were sacrificed at four time points: Zeitgeber Time 0, 6, 12, and 18. There are five replicates per condition.
Project description:Most metabolic studies are conducted in male animals; thus, the molecular mechanism controlling gender-specific pathways has been neglected, including sex-dependent responses to peroxisome proliferator-activated receptors (PPARs). Here we show that PPARalpha has broad female-dependent repressive actions on hepatic genes involved in steroid metabolism and inflammation. In males, this effect is reproduced by the administration of synthetic PPARalpha ligand. Using the steroid hydroxylase gene Cyp7b1 as a model, we elucidated the molecular mechanism of this PPARalpha-dependent repression. Initial sumoylation of the ligand-binding domain of PPARalpha triggers the interaction of PPARalpha with the GA-binding protein alpha bound to the target promoter. Histone deacetylase is then recruited, and histones and adjacent Sp1-binding site are methylated. These events result in the loss of Sp1-stimulated expression, and thus the down-regulation of Cyp7b1. Physiologically, this repression confers protection against estrogen-induced intrahepatic cholestasis, paving the way for a novel therapy against the most common hepatic disease during pregnancy. Experiment Overall Design: Expression profile difference between male and female PPARalpha wild-type and knock-out mice in liver and heart (3 pools of 4 animals in each group). Wild-type (12 males and 12 females) and knock-out PPARalpha SV129 mice (12 males and 12 females) approximately 10 to 12 weeks of age were killed at ZT14 and their livers and hearts quickly removed and frozen.
Project description:B cells encounter antigen to activate and then differentiate into plasma cells. Both multiple myeloma (MM) and some autoimmune diseases such as multiple sclerosis (MS) and systemic lupus erythematosus (SLE) are characterized with abnormal production of plasma cells. In both diseases, the process of B cells differentiate into plasma cell is disordered. To explore the novel therapeutic target to the process from naïve B cells to plasma cells via activated B cells, we determined the gene expression profile in activated B cells by affymetrix microarrays. Splenic activated CD5+B cells were sorted from 7-9-week female C57BL/6 mice by FACS and from EAE (MOG-induced chronic experimental allergic encephalomyelitis (EAE) in C57BL/6 mice is an animal model for MS) by CD19 microbeads, respectively. The transcripts in B cells were determined by Affymetrix Microarrays.
Project description:If the function of the nuclear receptor PPARa is well-known during a prolongated fasting, its hepatic biological function during feeding and refeeding conditions still needs to be investigated. Moreover, in vivo data collected so far on PPARa function during fasting were obtained using the total Ppara KO transgenic mouse model. To identify genes whose expression is under the strict dependence of hepatic PPARa activity, we generated a new mouse strain of PPARa-specific deletion in hepatocyte (albumin-Cre+/- Pparaflox/flox or LKO) and we compared them to total Ppara KO (KO), wild-type (WT) and liver WT (albumin-Cre-/- Pparaflox/flox or LWT) mice under three nutritional challenges. We used microarrays to detail the global programme of gene expression in liver of Ppara LKO, LWT, Ppara KO and WT male mice fed ad libitum, fasted for 24 hours and refed. There are 52 liver samples, each from an individual mouse. The samples are from Ppara liver KO (LKO), Ppara KO (KO), wild-type (WT) and liver WT (LWT) male mice of 8 week-old from the same genetic background (C57Bl/6J) fed ad libitum, fasted for 24 hours, fasted for 24 hours and then refed 24 hours more with glucose added in water (200g/l). In fed condition (Fed), n= 3 mice for LKO, LWT genotypes, n= 5 for KO and n= 4 fot WT; in fasting condition (Fas), n=5 for LKO, LWT and WT genotypes and n= 3 for KO; in refeeding condition (Ref), n= 5 for LKO, KO and WT genotypes and n= 4 for LWT. All mice were sacrified at ZT14.
Project description:Fenofibrate is a specific agonist of the nuclear receptor PPARa. To identify the gene expression under the strict dependence of hepatic PPARa activity, we generated a new mouse strain of PPARa-specific deletion in hepatocyte (albumin-Cre+/- Pparaflox/flox or LKO) and we compared them to total Ppara KO (KO), wild-type (WT) and liver WT (albumin-Cre-/- Pparaflox/flox or LWT) mice. We used microarrays to detail the global programme of gene expression in liver of Ppara LKO, LWT, Ppara KO and WT male mice. There are 36 liver samples, each from an individual mouse. The samples are from Ppara liver KO (LKO), Ppara KO (KO), wild-type (WT) and liver WT (LWT) male mice of 14 week-old from the same genetic background (C57Bl/6J) treated with Fenofibrate (100 mg/kg/day) or vehicle (aqueous solution of gum Arabic 3%) by daily gavage for 10 days. n= 4 mice for LKO, LWT and WT genotypes treated with vehicle; n=3 for KO mice treated with vehicle; n=5 mice for LWT, LKO and KO genotypes treated with fenofibrate; n=4 WT mice treated with fenofibrate. All mice were sacrified at ZT14.
Project description:ADRs (adverse drug reactions) are one of the main reasons for treatment discontinuation or alterations in dose regimens in clinical settings. Chemotherapy-induced ADRs are common, especially gastrointestinal-induced ones. Within TransQST (transqst.org), one of the main goal is to improve the in vitro-in vivo translation in toxicological studies, as well as translation between non-clinical species (such as rat and mouse) and humans. This experimental setup aimed to investigate the injury dynamics underlying repeated exposure to two doses of 5-FU (20 and 50 mg/kg), sampled at multiple time points (6, 24, 96 and 144h). The 144h time point comprises of recovery samples (i.e., 48h following the last dosing with 5-FU). This dataset is part of the TransQST collection.
Project description:Purpose: Avian photoreceptors are a diverse class of neurons, comprised of four single cones, the two members of the double cone, and rods. Many distinctive features of photoreceptor subtypes, including spectral tuning, oil droplet size and pigmentation, synaptic targets and spatial patterning, have been well characterized, but the molecular mechanisms underlying these attributes have not been explored. Furthermore, the signaling events and transcriptional regulators driving the differentiation of these diverse photoreceptors are currently unknown. Methods: To identify genes specifically expressed in distinct chicken (Gallus gallus) photoreceptor subtypes, we developed fluorescent reporters that label photoreceptor subpopulations, isolated these subpopulations using fluorescence-activated cell sorting, subjected them to next-generation sequencing, and conducted differential expression analysis. Results: We identified hundreds of differentially expressed genes from photoreceptor subpopulations labeled with rhodopsin, red opsin, green opsin, and violet opsin reporters. These genes are involved in a variety of processes, including phototransduction, transcriptional regulation, cell adhesion, maintenance of intra- and extra-cellular structure, and metabolism. Of particular note are a variety of differentially expressed transcription factors, which may drive and maintain photoreceptor diversity, and cell adhesion molecules that may mediate spatial patterning of photoreceptors and act to establish retinal circuitry. Conclusions: These analyses provide a framework for future studies that will dissect the role of these various factors in the differentiation of avian photoreceptor subtypes. mRNA expression profiling of 5 pairs of photoreceptor subtypes isolated from chicken retinal explants, 3 replicates per sample
Project description:Demonstration of hematopoietic stem cells (HSCs) was first shown in the mouse and was dependent on recipient bone marrow (BM) to support in vivo multilineage hematopoietic reconstitution, thereby illustrating non-cell-autonomous requirements for HSC functions. Murine studies have defined microanatomic compartments in the BM comprised of osteoblasts, mesenchymal cells, subsets of vasculature, and innervating neural cells functioning as an HSC-supportive niche. Despite the potential clinical applications, analyses of putative HSCs in the BM of humans has not been examined. Here, using human bone biopsies, we provide evidence of HSC propensity to endosteal regions of Trabecular Bone Area (TBA). Independent of phenotypic definitions based on prospective isolation, functional studies indicate that human HSCs residing in the TBA of human and transplanted recipients had superior regenerative and self-renewal capacity and are molecularly distinct to those repopulating the Long Bone Area (LBA). Consistent with the non-cell-autonomous nature of HSC function, osteoblasts in the TBA possess unique characteristics and expressed a key network of factors including those involving Notch activity which could regulate TBA vs. LBA location of human HSCs in vivo. Our study illustrates that human-mouse xenografts provide a surrogate to indigenous human HSC in the BM, and demonstrates that BM architecture plays a critical role in defining functional properties of human HSCs. Lin- MNCs from human Umbilical Cord blood (CB) were injected via tail vein into sublethally irradiated NOD/SCID adult mice. After 10 weeks post-transplantation, engrafted-CB cells were sorted based on the co-expression of CD45 and CD34, and absence or presence of CD38 using a FACSAria II (BD). Total RNA from purified populations was extracted and amplified as described previously (Shojaei et al., 2005). Amplified-labeled RNA was hybridized to HG-U133Plus v2.0 chip.