There is now wide acceptance that molecules of the extracellular matrix (ECM) have a regulatory role in both health and disease. Despite this acceptance, genetically engineered mice are still not available for use as tools to study the role of the large chondroitin sulfate proteoglycan, versican, which has been implicated, along with its binding partner hyaluronan, in a wide variety of both acute and chronic inflammatory events. Our laboratories have been involved with the study of atherosclerosis and lung infection, and have more recently collaborated in studies of asthma and diabetes, all of which have a critical chronic immune response that is characterized by the increased expression and accumulation of versican. We now propose to develop two genetically engineered mice, essential tools to determine the role that versican plays in both health and disease. In the first aim, we will generate a mouse with floxed exons of versican (versicanfl/fl mice) such that when crossed with mice expressing a regulatable Cre recombinase, a knock-out of versican expression can be achieved by homologous recombination (versican-/- mice). A conditional knockout is required as disruption of the versican gene results in embryonic lethality. In the second aim we will generate a versican- reporter mouse model to determine the mechanisms controlling the expression of versican. We will generate a mouse with the versican promoter driving the expression of the fluorescent protein, tdTomato. To accomplish this we will first build a targeting vector where a CRE- tdTomato fusion protein is targeted to the versican gene. This targeting construct will then be used to develop the versican-reporter mouse (versicanTdTR/cspg2). We will then breed and characterize the versicanTdTR/cspg2 mice to show that the tdTomato correlates with the expression and accumulation of and versican. In the third aim the kinetics and role of versican expression in the pulmonary inflammatory response to gram-negative bacteria will be determine through the use of versican-tdTomato reporter mice and versican-/- mice. We believe that the successful completion of the proposed studies in Aim 3 will provide compelling evidence that versican plays a key role in the inflammatory response to lung infection. When available, these genetically engineered mice will be available for the study of a wide variety of pathologies and injuries in which versican has been implicated as a relevant factor, such as in spinal cord injury, carcinogenesis and metastasis, birth defects, heart valve defects and great vessel transposition, and wound repair. The unambiguous identification of a direct role for versican in specific inflammatory events will stimulate future therapeutic strategies to target versican as a means of treating chronic inflammation. PHS 398 (Rev. 11/07) Research Plan
This project proposes to develop a conditional knockout of the versican gene in the mouse, as well as a versican reporter mouse for use in future studies to explore the role of versican as a component of the extracellular matrix with which immune cells interact. We believe these models will have wide utility, not just in gaining new insights into the mechanisms of chronic inflammation, but also other fields in which versican is thought to play a role, such as spinal cord injury, cancer and birth defects.
