Highly specific, temporally controllable mouse genetic tools for investigating in

描述(申请人提供)：神经胶质细胞与神经元形成亲密接触，并在发育过程中帮助协调大脑连接，并促进成年后的神经元功能。因此，阐明神经胶质细胞在体内的功能具有重要意义。然而，用于研究两种主要胶质细胞类型--成熟星形胶质细胞和NG2细胞--的高度特异性和适应性的小鼠品系仍然缺乏。在这里，我们建议创造小鼠遗传工具，使人们能够以时间可控的方式探索这两种类型的神经胶质细胞的功能。建议的Tet-Off二元系统由两组小鼠组成：1)在特定类型的神经胶质细胞中表达TTA的驱动系；2)在Tet操纵子(Teto)控制下表达转基因以用于细胞消融或转录剪裁的效应系。该系统将允许人们挑选和选择单独的驱动器和效应器线来研究神经胶质生物学中的特定问题。当与现有的Cre系相结合时，交叉遗传学可以提供前所未有的细胞类型特异性来分析体内的神经胶质功能。尽管表面上看起来很简单，但泄漏、杂色/镶嵌和沉默转基因表达的问题可能会破坏这些努力。经过精心的准备，我们的实验室现在已经准备好迎接这一挑战。通过建立和优化小鼠基因镶嵌系统，我们在基于ES细胞的敲入策略方面积累了丰富的经验，我们将使用精心设计的靶向策略来确保转基因表达的真实性和完整性。同样重要的是，我们已经广泛咨询了神经胶质生物学和小鼠遗传学领域的领导者，并将把这些转基因定位到最有希望的基因组位置，以实现忠实和特异的转基因表达。我们相信，这项资助中描述的遗传工具将对研究神经胶质细胞在中枢神经系统中正常功能和疾病中的作用产生广泛的影响。例如，星形胶质细胞或NG2细胞中的RNA标记将允许人们研究这些细胞在整个正常发育过程中或在某些病理条件下的转录图景。细胞消融实验可以帮助揭示特定类型的胶质细胞在成年后发育和认知功能的大脑连接中的关键贡献。将细胞消融应用于脑肿瘤研究，将有助于确定有效治疗干预的重要细胞靶点。最后，从拟议的工作中学到的原则将为今后系统的成功扩展提供坚实的基础，以解决神经科学中各种令人兴奋的主题。

DESCRIPTION (provided by applicant): Glial cells form intimate contact with neurons and help orchestrate brain wiring during development and facilitate neuronal functions in adulthood. Therefore, it is important to elucidate glial functions in vivo. However, highly specific and adaptable mouse lines for studying two major glial cell types, mature astrocytes and NG2 cells are still lacking. Here we propose to create mouse genetic tools that would allow one to probe into the function of these two types of glial cells in a temporally controllable fashion. The proposed Tet-OFF binary system consists of two groups of mice: 1) driver lines that express tTA in specific types of glial cells; 2) effector lines that express transgenes for cell ablation or transcriptomic profiling under the control of Tet Operon (TetO). The system would allow one to pick and choose individual driver and effector lines to study a specific problem in glial biology. When combined with existing Cre lines, intersectional genetics can provide unprecedented cell type-specificity to analyze glial functions in vivo. Although apparently straightforward, problems of leakiness, variegation/mosaicism, and silencing of transgene expression could derail such efforts. With careful preparation, our lab is now ready to take on this challenge. With our extensive experience with ES cell-based knock-in strategy gained from creating and optimizing a mouse genetic mosaic system, we will use carefully designed targeting strategies to ensure the faithfulness and completeness of transgene expression. As importantly, we have broadly consulted with leaders in the field of glial biology and mouse genetics, and will target these transgenes into the most promising genomic loci for faithful and specific transgene expression. We believe that genetic tools described in this grant will have a broad impact on studying the roles of glial cells for normal functions and diseases in the central nervous system. For example, RNA tagging in astrocytes or NG2 cells will allow one to study the transcriptional landscape of these cells throughout the normal development or under certain pathological conditions. Cell ablation experiments could help reveal the critical contributions of specific glial cell types in brain wiring during development and cognitive functions in adulthood. Applied to brain tumor research, cell ablation will help identify important cellular targets for effective therapeutic intervention. Lastly, principles learned from the proposed work will provide a firm foundation for the successful expansion of the system in the future to address a diverse range of exciting topics in neuroscience.