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NOGGIN AND CHORDIN KNOCKDOWN IN DISTRACTION OSTEOGENESIS : IN VITRO STUDY
Presenter: Priscille Grenier-Vallee, MD
Co-Authors: Kasaai B; Moffatt P; Hamdy RC
McGill University Shriners Hospital for Children

Introduction: Bone distraction osteogenesis (DO) is a long process that often implies social, psychological and medical complications. Bone morphogenic proteins (BMPs), being one of the most powerful osteogenic growth factors, have been used in the past to accelerate the process. The rapid clearance of exogenous BMPs has lead the recent research toward increasing endogenous BMPs. We hypothesize that by inhibiting BMP antagonists Noggin and Chordin using RNA interference we may upregulate endogenous BMP expression and enhance osteogenesis.

Methods: MC3T3 cells were transduced with lentiviruses expressing various shRNAs targeting the mouse Noggin (5 shRNAs) and Chordin (3 shRNAs) genes. A negative control (1 shRNA) that did not target Noggin or Chordin was also used. At various time points after infection, levels of RNA expression for Noggin and Chordin were monitored through RT-PCR. Western blotting were performed on the cell extracts and culture media to verify the expression and secretion of Noggin and Chordin proteins. Cell extracts were also analyzed on day 2 and 4 after transduction for alkaline phosphastase activity which is a marker of osteogenic differentiation.††

Results: †On non-tranduced MC3T3 cells, the levels of RNA expression of Noggin and Chordin was at its highest level on Day 7 after transduction. At this timepoint, qRT-PCR analysis showed that the shRNAs were effective in knocking down Noggin and Chordin endogenous mRNA levels down to 10% and 17%, respectively, by the most potent of shRNAs tested compared to control. Western Blot analysis also corroborates that the respective shRNAs were effective in knocking down the Noggin and Chordin proteins. Specific activity of alkaline phosphatase was increased in MC3T3 cells stably expressing Noggin and Chordin shRNAs.

Conclusion: Western blot analysis, qRT-PCR and ALP assay are consistent with a successful knockdown of Noggin and Chordin. The second step of our project was to address our hypothesis with an animal model of distraction osteogenesis.


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