CORE--TRANSGENIC ANIMALS

这个核心是一个传统的核心，在使用转基因小鼠在体内验证抗镰刀策略方面具有额外的作用，转基因小鼠构成了我们最初提案中项目1的一部分。核心服务包括三项功能：1)确保阿尔伯特·爱因斯坦医学院的转基因和基因靶向设施，在那里，RL Nagel为实现项目1中概述的目标而设计的小鼠将在Eric Bouhassira的监督下使用由Core B提供的DNA结构。2)确定表达所需血红蛋白的创始人，对其进行鉴定，并将其培育成表达α/H/β/S的转基因小鼠和/或由RMCE(目标3中所述)、α-基因敲除、β-基因敲除和其他转基因和基因敲除获得的新小鼠，并将它们回交到C57BL/6上，用于项目1、2和4；3)为其他项目提供分析服务；蛋白质鉴定、定量(高效液相色谱、质谱分析)和红细胞鉴定。我们将利用我们现有的特征良好的模型来开发表征抗镰刀策略有效性的方法开发两种新的方法来评估镰刀鼠和使用抗镰刀珠蛋白的小鼠的缺氧：1)HIF-1α作为局部缺氧的测试手段。2)应用BOLD和血流敏感的MRI技术来监测转基因模型中脱氧血红蛋白的存在和血流的改善/受损。利用S+S+安的列斯小鼠的异常严重程度和低水平伽马的营救来帮助设计更有效的抗病策略。我们将利用项目2中描述的重组酶介导盒交换(RMCE)技术来产生一种全新的镰状细胞小鼠模型来测试抗镰状珠蛋白。转基因表达水平不同，但在小鼠的一生中在拷贝数量和染色质结构上具有可比性的新的镰状细胞小鼠模型将简化这些动物的使用。消除珠蛋白缺乏、地中海贫血和具有不同水平抗镰状珠蛋白表达的可比小鼠，将大大简化我们建议的干预导致的病理生理学变化的解释。使用这项技术，我们希望能够毫不含糊地确定我们计划在体内测试哪种抗镰状珠蛋白是最好的。

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.