Endogenous barcoding to reveal neural stem cell lineage

摘要 关于成人大脑的干细胞，有几种部分重叠和部分冲突的模型 维持他们的池，分裂，并产生神经元和神经胶质细胞。解决这些模式很重要 因为它们意味着对认知功能的不同长期后果，压力和 疾病，以及对治疗的反应。根据模型的不同，这些后果的范围从 干细胞库的持续支持及其产生神经元和神经胶质后代的能力 干细胞库耗尽并停止产生后代的能力。在一定程度上，关于 干细胞生命周期的基本方案由所采用方法的固有局限性来解释。 要研究这个问题，现在仅限于分裂事件的核苷酸标记、克隆分析或活的 观察。在这里，我们提出了内生条形码作为一种正交方法，并描述了 评估其用于研究干细胞和神经元和神经胶质细胞生成的可行性的实验。这 该方法基于Polylox，一种新的Cre重组酶驱动的DNA重组底物 进入小鼠的生殖系。Cre诱导9个独特的DNA元素随机重组，产生超过 支持Polylox等位基因重组的一百万个不同的密码和唯一的标记细胞 以及它们所有的后代。我们建议在成人神经干细胞中诱导重组事件 携带Polylox等位基因的复合系的海马体并确定其总的组成 后代。此外，我们建议将Polylox内源条形码方法与单一条形码方法相结合 细胞转录学，以确定单个条形码细胞的轮廓及其在轨迹上的位置 从干细胞到分化的神经元或神经胶质细胞。因此，在我们的第一个特定目标中，我们将生成多等位基因 携带Polylox转基因和干细胞转基因组合的转基因小鼠- 特异性Cre重组酶和谱系标记，然后将评估和分离海马细胞 携带详细信息的条形码，并推导出它们之间的关系。在我们的第二个具体目标中，我们将产生 携带Polylox条码盒的额外多等位基因系，应用重组诱导的内源 条形码，然后用单细胞转录分析和条形码分析相结合作为一种新的 确定单个神经干细胞分裂、分化和谱系的方法。两者都有 方法还将有助于解决有关模型的一些悬而未决或相互矛盾的问题 干细胞的维持以及神经胶质和神经元后代的分裂和生成。我们的探索者 该项目将在神经干细胞、神经元和神经胶质细胞的研究中引入一种新的模式，并将为 作为进一步研究发育中和成人干细胞生命周期动态调节的平台 神经系统。

ABSTRACT There are several models, partially overlapping and partially conflicting, of how stem cells of the adult brain maintain their pool, divide, and give rise to neuronal and glial cells. Resolving those modes is important because they imply different long-term consequences for the cognitive function, effects of stress and disease, and response to therapies. Depending on the model, these consequences range from the continuous support of the stem cell pool and their ability to generate neuronal and glial progeny to the exhaustion of the stem cell pool and cessation of the ability to produce progeny. Partially, the debate about the basic scheme of the stem cell life cycle is explained by inherent limitations of the approaches employed to study this issue, which are now limited to nucleotide labeling of division events, clonal analysis, or live observation. Here we propose endogenous barcoding as an orthogonal approach and describe experiments to assess its feasibility for studying stem cells and generation of neurons and glia. This approach is based on Polylox, a new Cre recombinase-driven DNA recombination substrate introduced into the mouse germline. Cre induces random recombination of nine unique DNA elements, creating over a million distinct codes and uniquely marking cells that have supported recombination of the Polylox allele and all of their progeny. We propose to induce the recombination events in neural stem cells of the adult hippocampus of compound lines carrying the Polylox allele and determine the overall composition of their progeny. Furthermore, we propose to combine the Polylox endogenous barcoding approach with single cell transcriptomics to determine the profiles of individual barcoded cells and their position on the trajectory from stem cells to differentiated neurons or glia. Thus, in our first specific aim we will generate multiallelic transgenic mouse lines carrying a combination of the Polylox transgene with transgenes for stem cell- specific Cre recombinase and for lineage markers and will then assess and isolate hippocampal cells carrying particulars barcode and deduce their relation. In our second specific aim, we will generate additional multiallelic lines carrying Polylox barcode cassette, apply recombination-induced endogenous barcoding, and then use single-cell transcription analysis combined with barcode analysis as a novel approach for determining division, differentiation, and lineage of individual neural stem cells. Both approaches will also help to resolve some of the unanswered or contradictory questions about the models of stem cell maintenance and division and generation of glial and neuronal progeny. Our exploratory project will introduce a new modality in the studies of neural stem cells, neurons, and glia and will serve as a platform for further studies of dynamic regulation of the stem cell life cycle in the developing and adult nervous system.