The mission of the Molecular Core of the Core Center for Musculoskeletal disease at Yale will be to support the creation and analysis of animal models relevant to musculoskeletal disease and to facilitate the introduction and use of a range of molecular genetic methods by member investigators. To this end, we will provide both services for a number of specialized methodologies and technical assistance in a variety of essential molecular techniques. A central goal will be to form an integrated network within the CCMD; the creation of animal models of gene dysregulation and the characterization of target gene expression be fully supported the Molecular Core, with further analysis both in vivo and in vitro provided by the Cell and Physiology Cores. Aim 1. To provide as services, a) the construction of transgenes and gene disruption vectors, and b) the identification of genetically altered or naturally occurring mutant mice. This will include conventional transgenes, inducible transgenes using the tetracycline transactivator system, conventional gene targeting vectors and conditional gene targeting vectors using the cre-loxP method. Genotyping will be carried out by PCR or other blot analysis of tail DNA. Aim 2. To provide as services, a) the localization of gene expression by in situ hybridization, and b) the quantitation of gene expression by RNase protection. This will include the dissection and fixation of tissues, embedding in paraffin or OCT, sectioning by microtome or cryostat, preparation of riboprobes or oligoprobes, hybridization, emulsion autoradiography and image analysis. RNase protection will provide a sensitive means of relative or absolute quantification of mRNA. Aim 3. To provide technical assistance for both basic and advanced methods in molecular biology, which will range from hands-on training at the bench to consultative functions, such as experimental design, data interpretation and troubleshooting. Supported methods will include Southern and Northern blotting; RNA and genomic DNA preparation and analysis; stable or transient cell transfection by calcium phosphate, liposome or electroporation; reporter gene analysis using CAT, luciferase or growth hormone; the construction of vectors for the expression of proteins in bacteria or in eukaryotic cell lines using dominant selectable markers; cDNA cloning using oligonucleotides deduced from peptide sequence expression cloning, cDNA and genomic library construction; and competitive PCR and quantitative RT-PCR.