The purpose of this application is the incorporation of a Transgenic Mouse Core in the renewal of Dr. Sergio Jimenez's Program Project grant. In this grant, Dr. Jimenez is joining with the Dermatology Animal Core to produce animal models of human disease and to use transgenic mice to study protein structure and function in bone and cartilage. The procedures used by the Core to generate transgenic mice are pronuclear injection and gene-targeting technologies. Specifically, it is proposed to generate mutations in the gene for the transcription factor, COMP, using gene-targeting procedures. Mutations will also be generated in the collagen type II gene (COL2A1) in order to study structure/function relationships of this collagen as it interacts with other proteins. For introducing the site-specific mutations into these genes, the double-replacement technique is proposed, as well as alternate mutagenesis strategies. Techniques are proposed to generate transgenic mice that will express a transgene in a temporal and spatial-specific manner using an inducible expression system such as the Tetracycline control system. It is proposed to utilize the collagen type II promoter to drive an inducible transcription factor of the Tet-ON system (rtTA) to generate tetracycline inducible expression of a mutated1(II) collagen gene. Other methods, which may be employed, are the generation of conditional null, or dominant negative, phenotypes utilizing the Cre/Lox system. No human patients will be involved in the Core's studies.
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