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

描述（由申请人提供）：该拨款提案主要针对广泛的挑战领域（06）使能技术。具体来说，该提案满足了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；验证研究大脑连通性的新方法。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，除了一些罕见的例外，细胞类型或区域特异性的“驱动因素”在哺乳动物模式生物小鼠中基本上是不可用的，这使我们能够解剖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尚未解决的挑战。我们将利用最近优雅的比较基因组学工作，其中一组人类增强子元件被确定在多个脊椎动物物种中高度保守。重要的是，这些元件可以在小鼠神经系统的高度特异性区域可重复地驱动报告基因表达。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，产生由这种保守和超保守的人类增强子驱动的区域特异性Cre/CreER转基因小鼠系。此外，我们还开发了一个高效的分离- Cre/CreER系统。该系统允许我们以组合维恩图的方式使用增强子元素来表达具有不同增强子的分裂Cre/CreER半部分，并且活性Cre/CreER仅在两个增强子的重叠区域中重构。通过这种方式，我们将开发在神经系统中表达模式极其有限的Cre/CreER系。有了这些急需的Cre/CreER细胞系，神经科学家将能够以前所未有的能力和准确性绘制和操纵老鼠的神经回路，并在我们对哺乳动物大脑的了解方面取得巨大飞跃。该提案旨在利用基因组科学和新型转基因技术的现有知识来催化基础神经科学的重大进展。我们将产生多个在超保守增强子元件控制下表达Cre/CreER或分裂Cre/CreER的转基因Cre/CreER小鼠系。通过这种方式，将开发出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，对于理解人类大脑以及回答基础和临床神经科学中许多长期存在的问题至关重要

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