Project description:In response to the cytokines, macrophage colony-stimulating factor and receptor activator of NF-kB ligand, monocyte precursors differentiate into bone marrow-derived macrophages (BMDMs) that ultimately fuse to form multi-nucleated osteoclasts, following a tightly controlled genetic program where specific sets of genes are differentially expressed. We used microarrays to examine the gene expression profile underlying mouse osteoclast differentiation.
Project description:Osteoclasts are multinucleated giant cells generated by the fusion of precursors in response to stimulation with macrophage colony stimulating factor (MCSF) and receptor activator of NF-kB ligand (RANKL). These cells are the only cells capable of resorbing bone. Tartarate-resistant acid phosphatase is an enzyme secreted by osteoclasts that acts in bone resorption. Mice that are deficient for TRAP have shorter bones and their osteoclasts have decreased resorption capacity. In this project, we will isolate bone marrow macrophages from wild type and TRAP deficient mice, and differentiate the cells in osteoclasts in vitro. RNA will be extracted from macrophages and from macrophages stimulated with RANKL for both mouse lines (n=3/group) yielding 4 groups: Group 1 – macrophages from wild type mice, Group 2 – osteoclasts from wild type mice, Group 3 – macrophages from TRAP deficient mice, Group 4 – osteoclasts from TRAP deficient mice. The differential gene expression will be analyzed by RNAseq.
Project description:To identify the microRNAs that are involved in osteoclastogenesis, microRNA expression profiles in mouse bone marrow macrophages (BMMs) stimulated with RANKL (BMOc) were compared with that of control untreated BMMs. These results provide insights into the mechanisms to regulate osteoclastogenesis and bone resorption activities in osteoclasts by microRNA. BMMs were cultured with 20 ng/ml M-CSF in the presence or absence of 50 ng/ml RANKL for 24 hours. Cells were collected for total RNA isolation, and were subjected to microRNA array analysis.
Project description:Mouse bone marrow-derived OSCAR+CTR+ osteoclasts were harvested from mice with anterior cruciate ligament transection (ACLT) or sham operation by magnetic-activated cell sorting. The cells were lysated by Trizol reagent for total RNA extraction. The miRNA were profiled using the QuantiMir RT Kit (System Biosciences)
Project description:Osteoclastogenesis is induced by the stimulation of RANKL. In the early stage of osteoclast differentiation, the osteoclast progenitor cells are primed by M-CSF, following a tightly controlled genetic program where specific sets of genes are up-regulated by RANKL. Some of them, for instance, control differentiation, cell-cell fusion and bone resorption. We used microarrays to detail the global program of gene expression underlying osteoclastogenesis and identified various up-regulated genes during this process. Macrophages and osteoclasts were cultured for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain homogeneous populations of macrophages and osteoclasts in order to increase the temporal resolution of expression profiles. To that end, mouse bone marrow cells were cultured in the presence of M-CSF for three days and harvested as macrophage and oseteoclast common progenitor cells. Then common progenitor cells were further cultured in the presence of M-CSF alone for macrophages and M-CSF plus RANKL for osteoclasts, respectively.