The Francis Crick Institute.
MSD R&D Innovation Centre.
J Vis Exp. 2022 Oct 20(188). doi: 10.3791/64369.
Adult Hippocampal Neurogenesis (AHN), which consists of a lifelong maintenance of proliferative and quiescent neural stem cells (NSCs) within the sub-granular zone (SGZ) of the dentate gyrus (DG) and their differentiation from newly born neurons into granule cells in the granule cell layer, is well validated across numerous studies. Using genetically modified animals, particularly rodents, is a valuable tool to investigate signaling pathways regulating AHN and to study the role of each cell type that compose the hippocampal neurogenic niche. To address the latter, methods combining single nuclei isolation with next generation sequencing have had a significant impact in the field of AHN to identify gene signatures for each cell population. Further refinement of these techniques is however needed to phenotypically profile rarer cell populations within the DG. Here, we present a method that utilizes Fluorescence Activated Nuclei Sorting (FANS) to exclude most neuronal populations from a single nuclei suspension isolated from freshly dissected DG, by selecting unstained nuclei for the NeuN antigen, in order to perform single nuclei RNA sequencing (snRNA-seq). This method is a potential steppingstone to further investigate intercellular regulation of the AHN and to uncover novel cellular markers and mechanisms across species.
成人海马神经发生 (AHN) 是指在齿状回 (DG) 的颗粒下区 (SGZ) 内持续存在增殖和静止的神经干细胞 (NSC)，并将其分化为新生神经元，进而成为颗粒细胞层中的颗粒细胞，这一过程已在众多研究中得到充分验证。利用基因修饰动物，特别是啮齿动物，是研究调节 AHN 的信号通路以及研究组成海马神经发生龛的每个细胞类型的作用的有价值的工具。为了实现后者，将单细胞分离与下一代测序相结合的方法在 AHN 领域产生了重大影响，可用于鉴定每个细胞群体的基因特征。然而，为了对 DG 内更罕见的细胞群体进行表型分析，这些技术需要进一步细化。在这里，我们提出了一种方法，该方法利用荧光激活核分选 (FANS) 通过选择 NeuN 抗原未染色的核，从新鲜分离的 DG 中分离的单个核悬浮液中排除大多数神经元群体，从而进行单个核 RNA 测序 (snRNA-seq)。该方法是进一步研究 AHN 细胞间调节以及在不同物种中发现新的细胞标记物和机制的潜在垫脚石。
