Transgenic mice and bioinformatic tools to track astrocyte diversification insitu

描述(申请人提供)：这项建议的主要目标是产生新的生物信息学工具和小鼠转基因试剂，用于监测纹状体和杏仁核中星形胶质细胞的基因表达。这些实验旨在：(1)揭示移植的人类星形胶质细胞对植入的反应；(2)揭示移植的星形胶质细胞如何在小鼠Rett模型(MeCP2突变体)和焦虑模型(SERT突变体)中影响周围环境；(3)创造新的转基因动物，以表征星形胶质细胞的异质性和功能。现有的工具和方法不足以解决这些问题，部分原因是微阵列的动态范围和灵敏度有限，FAC分选和免疫扫描在纯化过程中有导致细胞变化的风险，而激光捕获需要大量的RNA扩增。两个目标将开发新的方法和试剂来解决这些限制。在目标1中，将优化一条生物信息学管道，以确定小鼠和人类细胞转运蛋白的来源。下一步，来自H9 ES细胞或Qthera人胎儿神经胶质限制前体(GRPs)的人类星形胶质前体细胞将被立体定向移植到小鼠的纹状体和杏仁核中，并允许其成熟。高含量的长阅读RNA测序和生物信息学将被用来解开人到老鼠移植的转录本的来源。这种特定物种的基因图谱数据应该为人类星形胶质细胞前体在纹状体和杏仁核中的行为提供新的信息。同时，将监测纹状体和杏仁核对移植细胞的反应。最后，将使用RNA测序技术检测星形胶质细胞如何响应和影响MeCP2和SERT突变小鼠的纹状体和杏仁核。星形胶质细胞前体细胞移植正在被考虑用于治疗精神疾病、疾病和中枢神经系统损伤的许多临床试验。这些生物信息学方法和新的数据集将有助于星形胶质细胞功能的进一步表征。在AIM中，将产生2个新的小鼠品系，以实现从异质脑组织中存在的星形胶质细胞亚型和线性相关神经元中选择性地提纯RNA。在星形胶质细胞启动子GFAP或神经元启动子突触素的控制下，将产生表达核糖体亲和标签Rpl22和荧光钙报告基因GCaMP3的转基因小鼠。这些记者将被限制在神经胶质细胞和神经元亚型，使用一种需要Cre重组的交叉方法。这些转基因基因将通过与祖细胞特异性和可诱导的Cre系杂交来表征，以激活胶质细胞亚群中的报告基因。与Rpl22结合的RNA将被分离和测序，以确定来自相同祖细胞的胶质细胞和神经元是否具有分子遗传学特征。这些新的小鼠品系应该对神经科学界具有广泛的价值，并有助于识别结合或区分星形胶质细胞亚群和来自共同祖先的神经元的分子特征。

DESCRIPTION (provided by applicant): The main goal of this proposal is to generate new bioinformatics tools and mouse transgenic reagents for monitoring astrocyte gene expression in the striatum and amygdala. These experiments are designed to: (1) reveal how transplanted human astrocytes react to engraftment, (2) reveal how transplanted astrocytes influence their surroundings in mouse Rett models (MeCP2 mutant) and anxiety models (SERT mutant), and (3) to create novel transgenic animals for characterization of astrocyte heterogeneity and function. The existing tools and methods to address these questions are inadequate, in part because the dynamic range and sensitivity of microarrays is limited, FAC-sorting and immunopanning risk causing cellular changes during the purification, and laser capture requires massive RNA amplification. Two Aims will develop new methods and reagents to address these limitations. In Aim 1 a bioinformatics pipeline will be optimized to identify the origin of transcipts from mouse and human cells. Next, human astrocyte progenitors derived from H9 ES cells or Qthera human fetal glial-restricted precursors (GRPs, "Q cells") will be stereotaxically transplanted into the striatum and amygdala of mice and allowed to mature. High content long-read RNA-sequencing and bioinformatics will be used to deconvolute the origin of transcripts from the human-into-mouse transplants. This species-specific gene profiling data should provide new information on how human astrocytes precursors behave in the striatum versus the amygdala. At the same time the response of the striatum and amygdala to the transplanted cells will be monitored. Finally, how astrocytes respond-to and influence the striatum and amygdala of MeCP2 and SERT mutant mice will be examined, using RNA-sequencing. Transplantation of astrocyte precursors is being considered for many clinical trials to treat mental illness, disease and CNS injury. These bioinformatics methods and new data sets should be useful for further characterization of astrocyte function. In Aim 2 novel mouse lines will be generated to enable the selective purification of RNAs from astrocyte-subtypes and lineally related neurons present in heterogeneous brain tissue. Transgenic mice will be created that express the ribosomal affinity tag Rpl22 and the fluorescent calcium reporter GCaMP3 under the control of the astrocyte promoter GFAP or the neuron promoter Synapsin. These reporters will be restricted to glial and neuronal subtypes using an intersectional approach that requires Cre-recombination. These transgenics will be characterized using crosses to progenitor-specific and inducible Cre lines to activate the reporters in subsets of glia. RNAs bound to Rpl22 will be isolated and sequenced to determine whether glia and neurons originating from the same progenitor cells share molecular-genetic features. These new mouse lines should have broad value to the neuroscience community and help to identify molecular features that either bind or distinguish subsets of astroctyes and neurons arising from shared progenitors.