AN INDUCIBLE MOLECULAR MEMORY SYSTEM TO RECORD TRANSIENT STATES OF CNS CELLS

 描述（由申请人提供）：大脑是一个非常复杂的器官，由数百种独特的细胞类型组成，这些细胞类型被组织起来形成复杂的神经回路。虽然我们在了解大脑功能和发育方面取得了进展，但显然还有很多东西需要学习。目前，所有可以用于大脑功能研究的全基因组方法都是破坏性的，这意味着当对一群细胞进行基因组分析时，细胞被破坏。这一事实限制了我们将大脑细胞中的早期分子事件与后来的行为或细胞变化联系起来的能力。例如，目前不可能将神经元中的转录变化与细胞是否成功并入记忆痕迹的知识联系起来。类似地，将神经元祖细胞中发生的早期分子事件与细胞做出的最终细胞命运决定联系起来是不可行的。我们已经着手开发一种变革性的技术，可以在分子事件发生时记录它们，然后可以在任何确定的时间段后读出。我们有一种称为转座子“名片”的新技术，在培养中，它为细胞提供了在特定时刻发生的蛋白质-DNA相互作用的分子记忆。在这里，我们建议适应这种技术在体内使用，使分子事件的回顾性基因组分析。我们将通过完成现有方法无法完成的四个测试用例实验来演示该技术的实用性。具体来说，我们将通过以下方式测试该方法：1）回顾性鉴定控制CNS中细胞类型特化的候选转录因子2）鉴定区分体内抗神经变性的神经元的特征，3）鉴定在小鼠发声行为期间变得活跃的神经元，同时定位这些神经元中活性依赖性转录因子的全基因组结合，以及4）鉴定区分被并入恐惧记忆痕迹的神经元与未被并入的神经元的分子特征。

 DESCRIPTION (provided by applicant): The brain is a remarkably complex organ comprised of hundreds of unique cell types that are organized to form sophisticated neural circuits. Although we have made progress toward understanding brain function and development, it is clear there is still much to be learned. Currently, all genome-wide methods that could be brought to bear on functional studies of the brain are destructive, meaning that as a genomic analysis is performed on a population of cells, the cells are destroyed. This fact limits our ability to connec early molecular events in the cells of the brain with later behavioral or cellular changes. For example, it is currently impossible to connect transcriptional changes in a neuron with knowledge of whether or not the cell was successfully incorporated into a memory trace. Similarly, it is not feasible to connect the early molecular events that occur in a neuronal progenitor cell with the final cell fate decision made by the cell. We have set out to develop a transformative technology that can record molecular events at the time that they occur and can then be read out later after any defined period of time. We have a novel technology called transposon `Calling Cards' that, in culture, provides cells with a molecular memory of protein-DNA interactions that occur at a particular moment in time. Here, we propose to adapt this technology for use in vivo enabling a retrospective genomic analysis of molecular events. We will demonstrate the utility of this technology by completing four test-case experiments that cannot be done with existing methods. Specifically, we will test the method by: 1) retrospectively identifying candidate transcription factors that control the specification of cell types in the CNS 2) identifying features that distinguish neurons resistant to neurodegeneration in vivo, 3) identifying the neurons that become active during mouse vocalization behavior while simultaneously mapping the genome-wide binding of activity-dependent transcription factors in these neurons, and 4) identifying the molecular features that distinguish neurons that were incorporated into a fear memory trace from those that were not incorporated.