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的星形胶质细胞基因表达。这些实验旨在：（1）揭示移植的人星形胶质细胞如何对植入反应，（2）揭示移植的星形胶质细胞如何影响小鼠Rett模型（MeCP 2突变体）和焦虑模型（SERT突变体）中的周围环境，以及（3）创建新型转基因动物以表征星形胶质细胞异质性和功能。解决这些问题的现有工具和方法是不够的，部分原因是微阵列的动态范围和灵敏度有限，FAC-sorting和免疫淘选在纯化过程中有引起细胞变化的风险，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。 在目标1中，将优化生物信息学管道，以识别来自小鼠和人类细胞的transcipts的来源。接下来，将来源于H9 ES细胞或Qthera人胎儿胶质限制性前体（GRP，“Q细胞”）的人星形胶质细胞祖细胞立体定向移植到小鼠的纹状体和杏仁核中并使其成熟。将使用高含量长读RNA测序和生物信息学来解卷积来自人到小鼠移植的转录物的来源。这种物种特异性基因分析数据应该提供关于人类星形胶质细胞前体在纹状体与杏仁核中的行为的新信息。同时，将监测纹状体和杏仁核对移植细胞的反应。最后，星形胶质细胞如何响应和影响MeCP 2和SERT突变小鼠的纹状体和杏仁核将使用RNA测序进行检查。星形胶质细胞前体的移植正在考虑用于许多临床试验，以治疗精神疾病，疾病和中枢神经系统损伤。这些生物信息学方法和新的数据集应该是有用的进一步表征星形胶质细胞的功能。 在目标2中，将产生新的小鼠系，以能够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纯化RNA。将产生在星形胶质细胞启动子GFAP或神经元启动子突触蛋白的控制下表达核糖体亲和标签Rp 122和荧光钙报告基因GCaMP 3的转基因小鼠。这些报告将被限制到胶质细胞和神经元亚型使用交叉的方法，需要Cre重组。这些转基因将使用与祖细胞特异性和诱导型Cre系的杂交来表征，以激活神经胶质亚群中的报告基因。将与Rp 122结合的RNA分离并测序以确定源自相同祖细胞的神经胶质和神经元是否共享分子遗传特征。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来自共同祖细胞的星形细胞和神经元子集的分子特征。

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.