Burden-2R01NS036193-06A1. Mus musculus

These experiments are designed to discover genes that are expressed selectively by synaptic nuclei in skeletal muscle with the particular goal of identifying genes that regulate motor axon growth and differentiation. We plan to isolate RNA from the dissected synaptic region of skeletal muscle and from the non-synaptic region of skeletal muscle and to identify the genes that are expressed at higher levels in the synaptic than non-synaptic region. Previously, we showed that motor axons fail to stop and differentiate in mice lacking MuSK, a receptor tyrosine kinase that is activated by motor neuron-derived Agrin. We hypothesize that MuSK activation normally leads to the production of a retrograde stop/differentiation signal that is encoded by a gene that is expressed preferentially in synaptic nuclei. In the absence of MuSK signaling, the retrograde signaling is not produced by synaptic nuclei, and consequently motor axons wander aimlessly over the muscle. We obtain 6 to 8 micrograms of total RNA from the dissected synaptic or non-synaptic region from a single P21 mouse diaphragm muscle. This is a standard procedure in the lab, and we have used these methods to analzye gene expression and to generate high-quality cDNA libraries. Because the synaptic zone is narrower in the left hemi-diaphragm, we will isolate RNA from this half of the diaphragm. In order to isolate sufficient RNA (5 micrograms from each sample), we will pool the synaptic and non-synaptic regions from two hemi-diaphragms. In order to reduce experimental variability, we wish to analzye expression in six samples: three samples of synaptic RNA and three samples of non-synaptic RNA. We will ship the isolated RNA samples to the Consortium in order to generate labeled cDNA, to screen Affymetrix mouse oligo arrays and to assist in the analysis. Several genes, including the subunits of the acetylcholine receptor, MuSK, acetylcholinesterase, and utrophin are known to be expressed preferentially in synaptic nuclei; thus, these genes serve as internal controls for the reliability and effectiveness of the screen. Most other genes, several of which we have analyzed in previous studies, including actin, GAPDH, runx1, nogoC, creatine kinase, etc. are expressed uniformly in skeletal muscle; thus, expression of these genes should be equally represented in synaptic and non-synaptic regions. Keywords: other Overall design: as above

本实验旨在筛选骨骼肌突触核选择性表达的基因，核心目标为鉴定调控运动轴突生长与分化的基因。
我们计划从解剖获取的骨骼肌突触区域与非突触区域中分离RNA，并筛选在突触区域中表达水平显著高于非突触区域的基因。
我们此前证实，在缺失MuSK（一种可被运动神经元源性集聚蛋白（Agrin）激活的受体酪氨酸激酶）的小鼠中，运动轴突无法停止迁移并完成分化。
我们提出假说：正常情况下，MuSK激活会诱导产生一种逆行性停止/分化信号，该信号由在突触核中优先表达的基因编码。
当缺失MuSK信号通路时，突触核无法产生该逆行信号，进而导致运动轴突在肌肉表面无序游走。
我们可从单只出生后21天（P21）小鼠的膈肌解剖获取的突触或非突触区域中提取6~8微克总RNA。
该实验流程为本实验室标准操作，我们此前已利用此类方法开展基因表达分析并构建了高质量的cDNA文库。
由于左侧半膈肌的突触区域更窄，我们将从该侧半膈肌中分离RNA。
为获取足够的RNA（每份样本5微克），我们将合并两份半膈肌的突触区域与非突触区域样本。
为降低实验变异度，我们计划对6份样本进行表达分析：3份突触RNA样本与3份非突触RNA样本。
我们将把已提取的RNA样本寄送至该联盟（Consortium），以完成标记cDNA的制备、Affymetrix小鼠寡核苷酸芯片的筛选，并协助后续数据分析。
已知包括乙酰胆碱受体亚基、MuSK、乙酰胆碱酯酶（acetylcholinesterase）以及utrophin在内的多个基因可在突触核中优先表达，因此这些基因可作为本次筛选可靠性与有效性的内参对照。
其余多数基因（包括我们在既往研究中已分析的肌动蛋白（actin）、甘油醛-3-磷酸脱氢酶（GAPDH）、runx1、nogo-C以及肌酸激酶（creatine kinase）等）在骨骼肌中呈均匀表达，因此这些基因的表达水平在突触区域与非突触区域中应无显著差异。
关键词：其他 整体实验设计：如上所述