CORE--TRANSGENIC ANIMALS

核心——转基因动物

• 批准号: 6657112
• 负责人: Mary E Fabry
• 金额: $ 26.66万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6657112
• 关键词: animal breeding; animal colony; biomedical facility; biotechnology; gene targeting; gene therapy; genetically modified animals; hemoglobin; laboratory mouse; sickle cell anemia

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.

该核心是传统的核心，并且在使用转基因小鼠的抗镰状化策略的体内验证中具有额外的作用，所述转基因小鼠形成了我们最初提议中的项目1的一部分。核心服务包括三个功能：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，将在Eric Bouhassira的监督下，用核心B提供的DNA构建体生成设计用于实现RL内格尔在项目1中概述的目标的小鼠。2)鉴定表达所需血红蛋白的创始人，对其进行表征，并将其培育为我们的转基因小鼠表达α/H/β/S和/或RMCE创建的新小鼠（如目标3所述）、α-敲除、β-敲除和其他可用的转基因和敲除品系，并将它们回交到C57 BL/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（HPLC、质谱）和红细胞表征。我们将使用我们目前良好表征的模型来开发用于表征抗镰状化策略的功效的方法。开发用于评估镰状小鼠和具有抗镰状化球蛋白的小鼠中的缺氧的两种新方法：1）HIF-1 α作为测试局部缺氧的手段。2)应用BOLD和血流敏感MRI技术监测转基因模型中脱氧血红蛋白的存在和血流改善/受损。使用S+S+安的列斯小鼠的异常严重性及其通过低水平伽马的拯救来帮助设计更有效的抗镰状化策略。我们将利用项目2中描述的酶介导的盒交换（RMCE）技术来产生一种全新类型的镰状细胞小鼠模型来测试抗镰状球蛋白。新的镰状细胞小鼠模型具有可变的转基因表达水平，但在小鼠的一生中拷贝数和染色质结构相当，这将简化这些动物的使用。消除珠蛋白不足，地中海贫血，和具有不同水平的抗镰状球蛋白表达的可比小鼠将大大简化我们提出的干预措施导致的病理生理学变化的解释。使用这项技术，我们希望能够明确地确定我们计划在体内测试哪种抗镰状化球蛋白最好。