Cozzolino, Carmine (2009) Identification of genes involved in osteoblast differentiation with an shRNA-based approach. [Tesi di dottorato] (Unpublished)

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Mesenchymal stem cells (MSCs) derived from bone marrow and they can differentiate in a variety of cell types, including osteoblasts, adipocytes, chondrocytes, myoblasts, hepatocytes, and neural cells. For this reason they provide a promising role for developing cell-based therapy for degenerative diseases. Understanding the mechanisms behind MSC cell fate determination is not easy, because the molecular processes that drive differentiation are complex and poorly understood. So, also if in the last years many improvements have been done, some problems still remain. We planned to investigate on differentiation of MSC and in particular we wanted to focus our activities towards osteoblast differentiation; at this aim, we silenced specific mRNAs using a mouse shRNA library present in our institute, composed of at least two silencing constructs for each transcript, in a 96-well-plates-based screening strategy. In the first part of this study we set up the components and the experimental conditions to perform the screening. After, we proceeded with the screening and we were able to screen a part of the library. With this methodology we identified genes that are possible candidates to have a role in osteoblast differentiation. First of all, we made a Gene Ontology classification of these candidates using bioinfomatic tools and we identified genes involved in different processes and having different functions. Among the candidate genes, a big part is represented by genes whose function is still unknown, some of which represent putative novel transcription factors that we named ObI- (Osteoblast inducer-). In this study we focused our experiments on the first of these genes that we identified, ObI-1. We also considered genes whose function is known and that are involved in different processes but with a non described role in osteogenic differentiation. Among these genes we focused our attention on Serine racemase, which role in osteogenic differentiation was already suggested, but not largely demonstrated. For both the candidates that we treated in this study we carried out experiments to confirm the impairment in osteogenic differentiation as effects of their silencing and we made expression analyses in tissues and in our cells. Furthermore, as regards ObI-1 candidate, we performed a more deeply analysis due to characterization of his function.

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