Transgenic mice and bioinformatic tools to track astrocyte diversification insitu

描述（由申请人提供）：本提案的主要目标是生成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移植的反应；(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）中影响其周围环境；(3)创建新的转基因动物来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，facc分选和免疫计划在纯化过程中可能导致细胞变化，激光捕获需要大量的RNA扩增。Two Aims将开发新的方法和试剂来解决这些限制。在Aim 1中，将优化生物信息学管道，以鉴定来自小鼠和人类细胞的转录本的来源。接下来，来自H9 ES细胞或Qthera人类胎儿胶质限制性前体（GRPs，“Q细胞”）的人类星形胶质细胞祖细胞将被立体定向移植到小鼠纹状体和杏仁核中并使其成熟。高含量的长读rna测序和生物信息学将用于解开人类向小鼠移植的转录本的起源。这种物种特异性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体中的行为提供新的信息。同时，纹状体和杏仁体对移植细胞的反应将被监测。最后，我们将利用rna测序技术研究星形胶质细胞对MeCP2和SERT突变小鼠纹状体和杏仁核的反应和影响。星形胶质细胞前体移植正被考虑用于许多治疗精神疾病、疾病和中枢神经系统损伤的临床试验。这些生物信息学方法和新的数据集将有助于进一步表征星形胶质细胞的功能。在Aim 2中，将产生新的小鼠系，以便从异质脑组织中存在的星形细胞亚型和直系相关神经元中选择性纯化rna。在星形胶质细胞启动子GFAP或神经元启动子Synapsin的控制下，将构建表达核糖体亲和标签Rpl22和钙荧光报告基因GCaMP3的转基因小鼠。这些报告者将被限制在使用需要cre重组的交叉方法的胶质和神经元亚型。这些转基因将通过与祖细胞特异性和可诱导的Cre系杂交来激活胶质细胞亚群中的报告基因。与Rpl22结合的rna将被分离并测序，以确定来自同一祖细胞的胶质细胞和神经元是否具有相同的分子遗传特征。这些新的小鼠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来自共同祖细胞的星形胶质细胞和神经元亚群的分子特征。

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.