Project description:To screen mRNAs specifically regulated by mTORC1, a global mRNA expression profile in calvarial osteoblasts (OBs) from mice with or without OB-specific Tsc1 knockout was developed using microarray. Wild type (WT) or OB-specific Tsc1 knockout (KO) mice were sacrificed, with calvarial osteoblasts harvested and subjected to total RNA extraction.
Project description:Wnt signaling is a major regulator of osteoblast differentiation and function. To investigate how Wnt3a signaling regulates osteoblastic gene expression and to identify the role of Lrp5 and Lrp6 in mediating Wnt3a signaling in osteoblasts, neonatal calvarial osteoblasts isolated from C57Bl6 (WT) and osteoblasts lacking Lrp5 (Lrp5KO), Lrp6 (Lrp6KO) and, both Lrp5 and 6 (Lrp5/6KO) were treated with Wnt3a for 24 hours and gene expression changes were quantified by RNA-seq.
Project description:ChIP-seq was performed in triplicate on isolated tissues (kidney, thyroparathyroid (TPTG), peripheral blood monocytes) from hormone treated (vehicle, 1,25(OH)2D3, FGF23, or PTH) mice from wildtype (WT), M1-IKO, M21-IKO, VDR-KO, or Cyp27b1-KO mice as indicated in the sample listings. The pBMC samples were isolated from untreated mice and subsequently treated ex vivo in cell culture with the indicated concentrations of 1,25(OH)2D3 or forskolin. Studies are published under PMID 28808057 and PMID 31053643.
Project description:We utilized the Illumina MouseRef-8 gene expression technology to quantify differential gene expression between wildtype mice and mice with Osterix driven Cre conditional knockout of Hdac3 (Hdac3-CKO). We compared the RNA extracted from calvaria from 8 wildtype and 8 conditional knockout litter matched mice using two separate Illumina MouseRef-8 chips. Mice with exon 7 of Hdac3 flanked by loxP sites were crossed with mice expressing Cre driven by the Osterix promoter. RNA from 5 day old mouse calvarial explants (digested for 20 minutes with collagenase) was purified using TRIzol according to the manufacturerâs protocol (Invitrogen) and reverse transcribed using Qiagenâs Quantitect Reverse Transcription Kit. Two independent microarray experiments were performed; each experiment used RNA from four wildtype and four conditional knockout litter matched mouse calvaria.
Project description:Transciptome profiling of sorted naive CD4+ T cells from control or Creld1 conditional KO mice (CD4-Cre) both directly ex vivo and with or without stimulation with anti-CD3/anti-CD28 antibodies to study the impact of Creld1 on T cell biology
Project description:To screen mRNAs specifically regulated by mTORC1, a global mRNA expression profile in calvarial osteoblasts (OBs) from mice with or without OB-specific Tsc1 knockout was developed using microarray.
Project description:This RNA-seqexperiment was designed to find transciptional differences between wildtype and Themis2-deficient B cells either directly ex-vivo or stimulated for 6 h with various stimuli in vitro. It is part of a larger study on the function of Themis2 in B cells. Therefore splenic, live, B220+ CD93- IgM+ CD23+ follicular B cells were sorted by flow cytometry from wildtype or Themis2-deficient (Themis2KO/KO) C57BL/6J mice. Unstimulated samples were were lysed directly after the sort. Stimulated samples were stimulated in vitro for 6 h at 37 degree Celsius at a concentration of 3 million cells/mL with either 10 microgram/mL anti-IgM or 10 microgram/mL LPS or 1 microgram/mL CD40L with 0.1 microgram/mL IL-4 and then lysed. RNA was extracted using Trizol reagent (Life Technologies) and cleaned up using the RNEasy Mini Kit (Quiagen). Single end, unstranded, poly-A-enriched libraries were made using the TruSeq RNA sample preparation kit (Illumina). Samples were analysed with an Illumina HiSeq 2000, collecting 13.2 to 76.1 million reads of 75 bases per sample.
Project description:Arterial stiffness is a prevalent, independent cardiovascular risk factor, but the underlying mechanisms are not well understood. Wall shear stress and shear-sensitive genes may promote arterial stiffening through clinically important signaling pathways. Our goal was to identify how disturbed blood flow leads to arterial stiffness using the mouse partial carotid ligation model. Here we used our in vivo partial carotid ligation model to induce d-flow in the LCA while the contralateral RCA continues to experience stable laminar flow using the C57BL/6x129SvEv mice, TSP-1 knockout (KO), and C57Bl/6J mice. We compared these to aged (80 week) mice which had increased arterial stiffness due to aging. Changes in gene expression were identified using microarrays that were performed on the endothelial-enriched RNA isolated from the carotids exposed to stable flow (RCA) and compared to disturbed flow (LCA). Arterial stiffness was determined ex vivo by biaxial mechanical testing and in vivo by ultrasound techniques. Myointimal hyperplasia and immunohistochemistry were performed in sectioned carotid arteries. In vitro testing of signaling pathways utilized oscillatory and laminar wall shear stress. Human arteries were tested ex vivo to validate critical results found in the animal model.
