Cloning and Identification of Differentially Expressed Transcripts in Primary Culture of GABAergic Neurons
Cloning and Identification of Differentially Expressed Transcripts in Primary Culture of GABAergic Neurons
复制标题
DOI：
10.1023/a:1012317520937
复制
发表时间：
2001-10
影响因子：
4.4
通讯作者：
Zhi Li;Qiang-Bing Li;Chun Xiao Sun;L. Hertz;A. Yu
中科院分区：
文献类型：
--
作者：
Zhi Li;Qiang-Bing Li;Chun Xiao Sun;L. Hertz;A. Yu
基于RNA的任意引物聚合酶链反应（RAP-PCR）被用来鉴定从E16小鼠大脑皮层制备的大脑皮层神经元的原代培养物中的差异表达的转录本。已知在该培养制备物中发现的大多数神经元是GABA能的。采用不同的引物组合，对PCR产物进行聚丙烯酰胺凝胶电泳分离。通过银染色的可视化揭示了具有总共约200个转录物的高分辨率RNA指纹图案。回收6个差异表达的cDNA片段，克隆并测序。NCBI数据库检索结果显示，有6个克隆与已知基因和表达序列标签（EST）高度同源，且在发育过程中表达上调或下调。在这些克隆中，克隆3.1.7与小鼠Reelin（神经元迁移和定位相关蛋白）具有99%的序列同源性。克隆4.6.2与大鼠前原骨形态发生蛋白-3 mRNA同源性为91%。克隆6.10.2与一个新的钙非依赖性α-latrotoxin受体的孤儿基因有90%的同源性，该受体刺激突触前神经递质的释放。北方印迹分析证实克隆6.10.2在分化和发育阶段的神经元中从第2天到第7天表达上调。
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.