Scientists discover possible target for treating and preventing osteoarthritis PLOSONE
The main goal of this study was to determine if overexpressing RORβ in an osteoblast-like cell would inhibit the osteoblast phenotype while inducing a chondrogenic phenotype. Here we demonstrate that stable expression of the nuclear receptor RORβ in cultured cells results in alteration of a gene program that is supportive of chondrogenesis and protective against development of OA.
. PCR product was obtained with Maxime PCR Premix . pLPCX vector was digested and then dephosphorylated before ligation using the Rapid DNA Dephos & Ligation Kit . Ampicillin resistance clones were verified using a 5’ sequencing primer. Selected clone was linearized with AseI restriction digestion and dephosphorylated with Antarctic Phosphatase prior usage in transfection.
Human osteoblast-like MG-63 cells were maintained in EMEM with 10% heat-inactivated fetal bovine serum . Linearized hRORβ/pLPCX plasmid was transfected using lipid mediated transfection method . 0.5 ug/ml of Puromycin was added into complete media for selection of stable expressing hRORβ/pLPCX in MG-63 cells. A clone expression high levels of RORβ and a mock-vector clone were used for the following experiments.Total RNA was extracted using a Qiagen Kit-74106.
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