Molecular cloning of wild-type and mutant Osterix and its localization in MC3T3-E1 cells
Morcos, Joseph Mounir
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Bones are crucial organs in the human body - deviations from proper formation results in harmful and sometimes lethal effects. Thus, it is imperative that the regulatory factors of bone formation be investigated. One such factor is Osterix/SP7 (OSX), a Zinc-finger-containing transcription factor required for bone formation first determined in Osx null mice. In patients with mutant Osterix (MT-OSX), bone was malformed. Hence it is clear that wild-type (WT)-OSX and MT-OSX have different effects on the biological mechanisms involved in bone formation. This study aims to identify these differences at the cellular level by investigating the localization of WT-OSX and MT-OSX by confocal immunofluorescence microscopy. WT and MT forms of OSX were first cloned into pcDNA3-FLAG tagged vectors. Over expression of WT-OSX and MT-OSX were observed and indicated that both WT-OSX and MT-OSX (frameshift mutation at c.1052) experienced post translational modifications. In order to visualize the localization of WT-OSX and MT-OSX in the cell, pcDNA3-FLAG-WT-OSX and pcDNA3-FLAG-MT-OSX were transfected into MC3T3-E1 cells. Cells were stained based on immunofluorescence methods and it was concluded that MT-OSX was unable to enter into some parts of the nucleus. Although the exact locations of WT-OSX and MT-OSX were not determined, there is a clear difference in the localization of WT-OSX and MT-OSX in the cells; this ultimately affects the transcriptional properties of MT-OSX which inhibits proper bone formation.
PLEASE NOTE: This work is protected by copyright. Downloading is restricted to the BU community: please click Download and log in with a valid BU account to access. If you are the author of this work and would like to make it publicly available, please contact firstname.lastname@example.org.Thesis (MSD) --Boston University, Henry M. Goldman School of Dental Medicine, 2013 (Department of Periodontology and Oral Biology).x, 47 leaves : ill.Includes bibliographic references: leaves 39-44.
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