Functional Genomics and Phenotyping Core (FGPC) Project Summary Phenotypic characterization of animal models with induced genetic mutations is the most robust way to elucidate the in vivo functions of genes and the role of mutations in the pathogenesis of diseases. To date, generation of human disease models in mice has been facilitated by embryonic stem (ES) cell technology, routine transgenesis and mutagenesis. Our laboratory has had over 20 years of experience generating mice using ES cell as well as pronuclear injection. In this PPG, we propose to adopt the simple, fast and highly efficient genome editing tool CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system for RNA-guided DNA editing in a combined Mouse Mutagenesis Subcore. We will generate mice with null, point mutations, and conditional alleles to mimic genetic variants of Osteogenesis Imperfecta (OI) found in patients and/or generate alleles to test specific hypotheses related to the Projects in this Program Project Grant (PPG). Utilizing these mouse models, we will examine how variants contribute to quantity and quality of skeletal tissue by examining bone geometry, microarchitecture, and material composition. Importantly, the Core will leverage the infrastructure, expertise, and resources of the Baylor College of Medicine Knockout Mouse Phenotyping Project (KOMP2) (see Overview B.6.e) combined with the infrastructure of the Texas Medical Center (TMC) Bone Disease Program of Texas (BDPT) to achieve cost effectiveness and throughput (see Overview B.6.d). We will also establish a Mouse Skeletal Phenotyping Subcore. This sub-core will perform in phenotyping including microCT imaging, bone histomorphometry, and biomechanical and material study. To achieve this cost effectively, it will also leverage the infrastructure of the TMC BDPT (see Overview B.6.d). The Mouse Skeletal Phenotyping Subcore consists of two components. The MicroCT Imaging component will quantify and assess bone microarchitecture using standard resolution microCT imaging. In addition, the X-radia microCT system combines microCT with phase contrast optics to achieve ultra-high resolution of bone and other soft tissues. The Biomechanical and Tissue Property Analysis component will perform biomechanical assessments of bone, tendon and ligament strength and viscoelastic parameters to determine the material factors that contribute to tissue strength. The combination of these assessments will allow us to fully characterize the strength, plastic and viscoelastic properties of the tested tissues. Bone strength is also governed by the bone matrix and its mineral composition, which are altered in aging and disease. Raman spectroscopy (RS) will be utilized to analyze the content of bone mineral and organic matrix, which allows for concurrent evaluation of the material composition as well as mineral-matrix interactions. With these analyses, quantitative and qualitative information of the bone composition will be obtained. Collectively, these cores serve critical functions by integrating the generation of mouse models of human bone disease with providing systematic phenotype analyses.
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