Genome-wide transcriptome analysis of neuron-enriched DRG cultures exposed to Wnt3a
ABSTRACT: To test the potential of canonical Wnt signalling to modulate nociception via transcriptional regulation in dorsal root ganglion (DRG) neurons, we performed a genome-wide transcriptome analysis of neuron-enriched DRG cultures treated with Wnt3a or vehicle for 6 hours. Total RNA obtained from neuron-enriched mouse DRG culture subjected to Wnt3a treatment for 6 hours was compared to a matched control (vehicle-treated) DRG culture.
Project description:Analysis of changes on gene expression levels in brain and bone for the linkage to phenotypical alterations found in behavior, neurology, nociception, bone remodelling and development. Total RNA isolated from bone and brain of Ali35 mutant mice compared to wildtype controls
Project description:Identification of newer compounds to modulate dendritic cell functions. Total RNA obtained from bone marrow-derived dendritic cells treated for 6 hours with small chemical compounds or vehicle alone in the presence or absence of lipopolysaccharide (LPS).
Project description:Local protein synthesis in sensory neuron axons is necessary for axonal regeneration with the efficiency of regeneration decreasing with age. Because the full repertoire of transcripts in embryonic and adult rat sensory axons is unknown we asked how the pool of mRNAs dynamically changes during ageing. We isolated mRNA from pure axons and growth cones devoid of non-neuronal or cell body contamination. Genome-wide microarray analysis reveals that a previously unappreciated number of transcripts are localised in sensory axons and that this repertoire changes during development toward adulthood. Embryonic sensory axons are enriched in transcripts encoding cytoskeletal-related proteins with a role in axonal outgrowth. Surprisingly, adult axons are highly enriched in mRNAs encoding immune molecules with a role in nociception. To validate our experimental approach we show that Tubulin-beta3 mRNA is present only in embryonic axons where it is locally synthesised. In summary, we show that the population of axonal mRNAs dynamically changes during development, which may partly contribute to the intrinsic capacity of axons at different ages to regenerate after injury and to modulate pain. Pure axonal RNA were extracted from the axons of embryonic and adult dorsal root ganglion neurons, each with 5 biological replicates. The axonal transcriptomes were analysed using Affymetrix Rat Genome 230 2.0 Arrays.
Project description:In order to examine the role of macrophage heparan sulfate proteoglycans in atherogenesis, we inactivated the biosynthetic gene N-acetylglucosamine N-deacetylase-N-sulfotransferase 1 (Ndst1) in macrophages. In order to determine the impact of altering Ndst1 expression, we crossbred mice bearing a conditional loxP-flanked (“floxed”) allele of Ndst1 (Ndst1f/f) to transgenic mice expressing the bacterial Cre recombinase under control of the lysozyme 2 promoter (LysMCre) to drive inactivation of the gene in myeloid cells. We compared the transcriptome of bone marrow derived macrophages from Ndst1f/fLysMCre- and Ndst1f/fLysMCre+ macrophages in basal growth medium and after foam cell conversion using 50µg/ml of aggregated LDL (agLDL) to asses the impact of altered macrophage heparan sulfate sulfation on gene expression. Total RNA obtained from bone marrow derived macrophages (BMDM) derived form two wild-type and two Ndfst1-deficient mice before and after foam cell conversion and analyzed in diplicate (n=4 for each conditions; total of 4 conditions)
Project description:Analysis of gene expression differences in three types of mouse motor neurons: (1) those harvested from E13.5 mouse embryos; (2) those derived from embryonic stem cells by directed differentiation; and (3) those made from mouse embryonic fibroblasts by transcription factor reprogramming. Hb9::GFP+ mouse motor neurons were obtained by three methods [(1) dissection of Hb9::GFP-transgenic E13.5 mouse embryo spinal cord; (2) directed differentiation of an Hb9::GFP-transgenic mouse embryonic stem cell line, V6.5; (3) reprogramming of Hb9::GFP-transgenic mouse embryonic fibroblasts by overexpression of 10 transcription factors]. Each type of motor neuron was purified by FACS and harvested in Trizol. Total RNA was purified and prepared for hybridization onto Illumina MouseRef-8.
Project description:Analysis of rapamycin effects on white adipose tissue at gene expression level. The hypothesis tested in the present study was that rapamycin could modify immune cell composition and inflammatory state of the adipose tissue of obese mice. Total RNA prepared from the adipose tissue of obese mice treated with rapamycin or its excipient solution (referred as vehicle), compared to adipose tissue of lean mice.
Project description:Efforts to develop more effective therapies for acute leukemia may benefit from high-throughput screening systems that reflect the complex physiology of the disease, including leukemia stem cells (LSCs) and supportive interactions with the bone-marrow microenvironment. The therapeutic targeting of LSCs is challenging because LSCs are highly similar to normal hematopoietic stem and progenitor cells (HSPCs) and are protected by stromal cells in vivo. We screened 14,718 compounds in a leukemia-stroma co-culture system for inhibition of cobblestone formation, a cellular behavior associated with stem-cell function. Among those that inhibited malignant cells but spared HSPCs was the cholesterol-lowering drug lovastatin. Lovastatin showed anti-LSC activity in vitro and in an in vivo bone marrow transplantation model. Mechanistic studies demonstrated that the effect was on-target, via inhibition of HMG-CoA reductase. These results suggest that statins should be re-evaluated as anti-leukemia agents, and illustrate the power of merging physiologically-relevant models with high-throughput screening. Freshly isolated LSCe cells were treated with either 5µM BRD7116 or DMSO vehicle control for 6 hours in suspension in IMDM (12440053, Invitrogen), 10% FBS. Total RNA was extracted from the cells, labeled, and hybridized to MouseRef-8 v2.0 Expression BeadChips. Hartwell, K.A. et al., In Press (full citation forthcoming)
Project description:We investigated the role of breast cancer stem-like cells (CSCs-like), isolated from primary tumor, in promoting bone metastases in a human-in-mice model. Luciferase-transduced CD44+CD24- breast CSCs-like were injected through subcutaneous (SC) and intracardiac (IC) route in nonobese/severe combined immunodeficient (NOD/SCID) mice carrying subcutaneous human bone implants. The implanted bone was viable, active and human neo-vascularization was present. By in vivo luciferase imaging, we monitored tumor growth and detected bone-localized breast CSCs-like, both after SC and IC injection. Bone metastatic lesions were histologically evident, and tumor cells expressed epithelial markers and vimentin. Bone metastatic cells isolated from bone implants showed a CD44-CD24+ phenotype and re-created tumors and bone metastases after injection in secondary mice. A “bone tropism” expression signature was found to distinguish bone-colonizing cells from parental CSCs-like and to persist at subsequent passages also in the absence of surrounding bone tissue. The bone tropism signature displayed significant enrichment in genes discriminating bone metastases of breast cancer from metastases at other organs. Our results demonstrate the ability of breast CSCs-like to promote bone metastasis and provide a CSCs-like bone tropism signature, with potential prognostic value. C10 breast cancer stem-like cells (CSCs-like) were derived as mammospheres from a lobular-infiltrating breast carcinoma (ER+, HER2-). Samples are organized in the following groups: (i) Breast CSCs-like (C10, duplicate); (ii) Luciferase-transduced CSCs-like (C10L, simplicate); (iii) Bone-isolated C10L metastatic cells (C10-bone, duplicate) and subsequently (iv) grown in vitro as spheroids (C10-CSC, simplicate) or (v) re-grown in subcutaneous implants (C10-SC, duplicate).
Project description:HumanHT-12v4 microarray of cells in proliferation and differentiation conditions treated with or without 2 uM retinoic acid Cells were treated with or without 2 uM retinoic acid, in proliferation (KSFM + 5 ng/ml EGF + 50 ug/ml bovine pituitary extract) and differentiation (DMEM/F12 + 10 ng/ml EGF + 10% FBS) media