Developing drivers for neuron type-specific gene expression

 描述(申请人提供)：将Cre蛋白定向到特定神经元类型的驱动程序系已被证明是非常有价值的工具，不仅可以可视化特定的神经元类型，还可以通过Cre介导的光遗传探针的激活来操纵它们的活动，或者通过Cre介导的基因敲除来评估基因功能。大多数Cre驱动系，如基于BAC的Cre驱动或将Cre敲入特定的基因座，监测整个遗传座位的完整表达模式。然而，很少有基因是在特定神经元类型的非常小的群体中唯一表达的，这种细胞特异性的缺乏限制了这些驱动系的使用。我们建议开发转基因小鼠驱动系，将Cre基因的表达定向到小鼠大脑不同区域中数量非常有限的神经细胞类型，从而提供工具来精确绘制它们的功能和分子组成。为了实现这一目标，我们的目标是检验根据我们过去在线虫中的工作建立的假设，即小鼠基因座的顺式调控元件编码脊椎动物中枢神经系统中四个主要神经递质系统的囊泡转运体，谷氨酸、氨基丁酸(GABA)和乙酰胆碱(ACh)是由单个高度细胞类型特异性的顺式调控元件组成的模块组装。我们将在实验中测试这一假设，即单个独立的顺式调节元件的表达可能会将胆碱能、谷氨酸和GABA能区细分为小鼠中枢神经系统的有限区域，从而构成可重复性和高度特异性的驱动因素，以指导允许神经元和神经元电路的基因操作的基因表达。这种顺式调控解剖方法可以解决目前大多数可用的驱动器线不能唯一地针对有限数量的细胞的特异性问题。

 DESCRIPTION (provided by applicant): Driver lines that direct Cre protein to specific neuron types have proven to be invaluable tools to not only visualize specific neuron types but also to manipulate their activity through the Cre- mediated activation of optogenetic probes or to assess gene function by Cre-mediated gene knockout. Most Cre driver lines, such as BAC-based Cre drivers or knock-ins of Cre into specific loci, monitor the complete expression pattern of entire genetic loci. However, very few genes are exclusively expressed in very small populations of specific neuron types and this lack of cellular specificity limits the use of these driver lines. W propose here to develop transgenic mouse driver lines that direct Cre expression to very restricted numbers of neuronal cell types in different regions of the mouse brain, thereby providing tools to precisely map their function and molecular composition. To achieve this aim, we aim to test the hypothesis - built from our past work in the nematode C.elegans - that the cis-regulatory control elements of the mouse loci that encode the vesicular transporters for the four main neurotransmitter systems in the vertebrate central nervous system, glutamate and -aminobutyric acid (GABA) and acetylcholine (ACh) are composed of a modular assembly of individual, highly cell type-specific cis-regulatory elements. We will experimentally test the hypothesis that the expression of individual, isolated cis-regulatory elements may subdivide cholinergic, glutamatergic and GABAergic domains into restricted and perhaps novel domains of the mouse central nervous system and thereby constitute reproducible and highly specific drivers for directing the expression of genes that allow the genetic manipulation of neurons and neuronal circuits. This cis-regulatory dissection approach may solve the specificity problem of most currently available driver lines that are unable to exclusively target restricted numbers of cells.