An optogenetic toolkit for the interrogation and control of single cells.

 描述（由申请人提供）：我们在细胞和回路水平上对脑功能的理解严重依赖于以高特异性询问和改变神经细胞的能力。通过单光子或多光子激发使用光是提供足够分辨率以在细胞和亚细胞水平探测大脑的唯一方法。虽然光激活分子，如光遗传蛋白或光笼化合物，已经在神经科学中获得了许多关键的见解，但它们的使用仍然限于那些可能受膜通道影响的过程。我们建议开发一个工具包，允许使用光来询问、调节和修改脑细胞中的遗传信息。我们将建立在我们最近开发的技术，“激光标签”，基于蛋白质标签（即SNAP标签或HALO标签）和笼状化学配体之间的光依赖性相互作用。这种相互作用可用于通过亲和纯化回收分子或迫使细胞内蛋白质的二聚化。将SNAP和HALO融合到不同的细胞组分将允许我们1）从单细胞中回收DNA和RNA用于下游分析; 2）通过将激活子和阻遏子结构域募集到特定的基因组位点来调节转录; 3）通过病毒感染以单细胞分辨率递送转基因。这些研究的整体将B一个广泛的新技术与潜在的转变我们的能力，以针对特定的细胞类型 用于基因和分子研究

 DESCRIPTION (provided by applicant): Our understanding of brain function at the cellular and circuit level is critically dependent on the ability to interrogate and alter neural cells withhigh specificity. The use of light, either through single-photon or multi- photon excitation, is the onl method that provides sufficient resolution to probe the brain at the cellular and subcellular levels. While light-activated molecules, like optogenetic proteins or photocaged compounds, have allowed many key insights in neuroscience, their use is still limited to those processes that can be affected by membrane channels. We propose to develop a toolkit allowing the interrogation, regulation, and modification of genetic information in brain cells using light. We wll build on a technology we have recently developed, "LaserTag", based on the light-dependent interaction between protein tags (i.e. SNAP-tag or HALO-tag) and caged chemical ligands. Such interaction can be either use to recover molecules through affinity purification or to force the dimerization of proteins within a cell. Fusing SNAP and HALO to different cellular components will allow us to 1) Recover DNA and RNA from single cells for downstream analysis; 2) regulate transcription by recruiting activator and repressor domains to specific genomic loci; 3) deliver transgenes through viral infection with single cell resolution. The overall of these studies will b a broad new technology with the potential of transforming our ability to target specific cell types in the brain for genetic and molecular studies.