This Core is a traditional Core and has an additional role in in vivo validation of anti-sickling strategies using transgenic mice that formed a part of Project 1 in our original proposal. The Core services consist of three functions: 1) Make sure of the Transgenic and Gene Targeting Facility at the Albert Einstein College of Medicine where mice designed to achieve the goals outlined in Project 1 by RL Nagel will be generated with DNA constructs supplied by Core B under supervision of Eric Bouhassira. 2) Identify founders expressing the desired hemoglobins, characterize them, and breed them to our transgenic mice expression alpha/H/beta/S and/or the new mice created by RMCE (described in Aim 3), alpha-knockout, beta-knockout, and other transgenic and knockout lines available and backcross them onto C57BL/6 for use in Projects 1, 2, and 4, 3) Provide analytic services for other projects; protein identification, quantification (HPLC, mass spec), and red cell characterization. We will use our current well characterized models to develop methodologies for characterizing the efficacy of anti-sickling strategies Develop two new methods for evaluating hypoxia in sickle mice and those with anti-sickling globins: 1) HIF-1 alpha as a means of testing for local hypoxia. 2) Apply BOLD and flow sensitive MRI techniques to monitor for the presence of deoxyhemoglobin and improved/impaired flow in transgenic models. Use the anomalous severity in the S+S+Antilles mouse and its rescue by low levels of gamma to help design more effective anti-sickling strategies. We will take advantage of the Recombinase Mediated Cassette Exchange (RMCE) technology described in Project 2 to generate a completely new type of sickle cell mouse model to test anti-sickling globins. New sickle cell mouse models with variable levels of expression of the transgene but which are comparable in copy number and chromatin structure over the lifetime of the mouse will simplify use of these animals. Elimination of globin insufficiency, thalassemia, and comparable mice with various levels of anti-sickling globin expression will considerably simplify the interpretation of changes in pathophysiology resulting from our proposed interventions. Using this technology, we expect to be able to unambiguously determine which of the anti-sickling globins we are planning to test the best in an in vivo context.
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