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密切接触，在发育过程中帮助协调大脑线路，并促进成年期的神经元功能。因此，阐明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细胞的高特异性和适应性小鼠系仍然缺乏。在这里，我们建议创建小鼠遗传工具，使人们能够以一种暂时可控的方式探索这两种胶质细胞的功能。提出的Tet-OFF二元系统由两组小鼠组成：1)在特定类型的胶质细胞中表达tTA的驱动系；2)在Tet操纵子（TetO）控制下表达用于细胞消融或转录组谱的转基因的效应系。该系统将允许人们挑选单独的驱动和效应线来研究神经胶质生物学中的特定问题。当与现有的Cre系结合时，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胶质细胞功能。虽然看起来很简单，但泄漏、杂色/镶嵌和转基因表达沉默等问题可能会破坏这种努力。经过精心准备，我们的实验室已经准备好迎接这一挑战。我们通过创建和优化小鼠遗传镶嵌系统获得了丰富的ES细胞敲入策略经验，我们将使用精心设计的靶向策略来确保转基因表达的准确性和完整性。同样重要的是，我们广泛咨询了神经胶质生物学和小鼠遗传学领域的领导者，并将这些转基因靶向到最有希望的基因组位点，以实现忠实和特异性的转基因表达。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景观。细胞消融实验可以帮助揭示特定的神经胶质细胞类型在大脑发育和成年期认知功能中的关键作用。应用于脑肿瘤研究，细胞消融将有助于识别重要的细胞靶点，进行有效的治疗干预。最后，从建议的工作中学到的原则将为将来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.