Li Z, Li Q, Sun C X, Hertz L, Yu A C
Brain Research Institute, Shanghai Research Center of Life Sciences, Chinese Academy of Sciences.
Neurochem Res. 2001 Oct;26(10):1101-5. doi: 10.1023/a:1012317520937.
A RNA based arbitrarily primed polymerase chain reaction (RAP-PCR) was used to identify differentially expressed transcripts in primary cultures of cerebral cortical neurons prepared from E16 mouse cerebral cortex. The majority of neurons found in this culture preparation are known to be GABAergic. Different primer combinations were used, and the PCR products were separated on PAGE. Visualization by silver staining revealed a high resolution RNA fingerprint pattern with a total of about 200 transcripts. Six differentially expressed cDNA fragments were recovered, cloned and sequenced. The results of a NCBI database search showed that 6 clones were highly homologous to known genes and expressed sequence tags (ESTs), and that they were either up-regulated or down-regulated during development. Among these clones, Clone 3.1.7 shared 99% sequence homology to mouse Reelin, a neuronal migration and positioning related protein. Clone 4.6.2 shared 91% homology to Rat prepro bone morphogenetic protein-3 mRNA. Clone 6.10.2 had 90% homology to a novel orphan gene of calcium-independent alpha-latrotoxin receptor, which stimulates presynaptic neurotransmitter release. Northern blot analysis confirmed the up-regulated expression profile of Clone 6.10.2 in neuron from Day 2 to 7 during stages of differentiation and development.
基于RNA的任意引物聚合酶链反应（RAP-PCR）被用于鉴定从E16小鼠大脑皮层制备的大脑皮层神经元原代培养物中差异表达的转录本。已知在这种培养物制备中发现的大多数神经元是γ-氨基丁酸能的。使用了不同的引物组合，并将PCR产物在聚丙烯酰胺凝胶电泳（PAGE）上分离。银染可视化显示出高分辨率的RNA指纹图谱，共有约200个转录本。回收、克隆并测序了六个差异表达的cDNA片段。美国国立生物技术信息中心（NCBI）数据库搜索结果表明，6个克隆与已知基因和表达序列标签（EST）高度同源，并且它们在发育过程中要么上调要么下调。在这些克隆中，克隆3.1.7与小鼠Reelin（一种与神经元迁移和定位相关的蛋白质）的序列同源性为99%。克隆4.6.2与大鼠前体骨形态发生蛋白-3 mRNA的同源性为91%。克隆6.10.2与一种新型孤儿基因——非钙依赖性α- latrotoxin受体的同源性为90%，该受体刺激突触前神经递质释放。Northern印迹分析证实了克隆6.10.2在分化和发育阶段第2天至第7天的神经元中表达上调。
