CNS Region Specific Cre/CreER Transgenic Mouse Lines Driven by Conserved Human En

描述(由申请人提供)：这项资助提案主要针对广泛的挑战领域(06)使能技术。具体地说，该提案满足这一主要挑战中的两个子领域：06-NS-105将重要技术引入神经科学；06-NS-106验证研究大脑连接的新方法。哺乳动物的神经系统在神经元的剪切数量(数十亿)和神经元亚型的多样性(数千)方面都非常复杂。然而，除了少数罕见的例外，在哺乳动物模式生物--小鼠中，基本上没有细胞类型或区域特定的“驱动因素”，这让我们能够剖析哺乳动物大脑的精确连接和功能。我们的建议旨在解决这一未得到满足的挑战。我们将利用最近优雅的比较基因组学工作，其中确定了一组在多种脊椎动物物种中高度保守的人类增强子元件。重要的是，这些元件可以在小鼠神经系统中高度特异的区域驱动报告基因的重复性表达。我们将利用从基因组科学中获得的令人兴奋的知识，利用一种新的高通量睾丸电穿孔方法，在这种保守和超服务的人类增强子的驱动下，培育出区域特异性的Cre/Creer转基因小鼠系。此外，我们还开发了一个高效的Split-Cre/Creer系统。这个创新的系统允许我们以组合维恩图的方式使用增强子元件来表达具有不同增强子的分裂的Cre/Creer半部分，并且活性Cre/Creer只在两个增强子的重叠结构域中重组。通过这种方式，我们将培育出在神经系统中表达模式极其受限的Cre/Creer系。有了这些亟需的Cre/Creer线，神经科学家将能够以前所未有的能力和准确性绘制和操作小鼠神经回路，并在我们对哺乳动物大脑的知识中获得巨大飞跃。健康相关性这项提议是利用基因组科学和新型转基因技术的现有知识，促进基础神经科学的重大进步。我们将建立多个Cre/Creer转基因小鼠系，其中Cre/Creer或Split Cre/Creer在超强启动子元件的控制下表达。这样，就可以建立在小鼠大脑特定区域具有高度限制性表达模式的Cre/Creer转基因株系。更全面地了解复杂的小鼠大脑网络的连通性和功能，对于理解人脑以及回答基础和临床神经科学中的许多长期问题将是至关重要的。

DESCRIPTION (provided by the applicant): This grant proposal primarily addresses broad Challenge area (06) Enabling Technologies. Specifically, the proposal meets two sub-areas within this main Challenge: 06-NS-105 Importing important technologies into neuroscience; 06-NS-106 Validating new methods to study brain connectivity. The mammalian nervous system is enormously complex in terms of both the shear number of neurons (billions) and the diversity of neuronal subtypes (thousands). However, with a few rare exceptions, cell type- or region-specific "drivers" are largely unavailable in the mammalian model organism, the mouse, that allow us to dissect the precise connections and functions of the mammalian brain. Our proposal is aimed at solving this unmet challenge. We will take advantage of the recent elegant comparative genomics work in which a set of human enhancer elements were identified that are highly conserved across multiple vertebrate species. Importantly, these elements can drive reporter expression reproducibly in highly specific regions in the mouse nervous system. We will utilize this exciting knowledge obtained from genome sciences to generate region-specific Cre/CreER transgenic mouse lines driven by such conserved and ultraconserved human enhancers using a novel high throughput testis-electroporation method. Moreover, we have also developed an efficient split- Cre/CreER system. This innovative system allows us to use the enhancer elements in a combinatorial Venn Diagram manner to express the split Cre/CreER halves with different enhancers, and the active Cre/CreER is only reconstituted in the overlapping domains of two enhancers. In this way, we will develop Cre/CreER lines with extremely restricted expression pattern in the nervous system. With these much-needed Cre/CreER lines, neuroscientists will be able to map and manipulate mouse neural circuits with unprecedented ability and accuracy and to gain quantum leaps in our knowledge about the mammalian brain. Health Relevance This proposal is to capitalize on existing knowledge from genome sciences and novel transgenic technologies to catalyze major progress in basic neuroscience. We will generate multiple transgenic Cre/CreER mouse lines in which Cre/CreER or split Cre/CreER is expressed under the control of ultraconserved enhancer elements. In this way, Cre/CreER transgenic lines with highly restricted expression pattern in specific regions of the mouse brain will be developed. More complete understanding of the connectivity and function of the complex mouse brain networks will be critical for understanding the human brain and for answering many longstanding questions in basic and clinical neuroscience