The role of astrocytes in maintaing reuronal function

本研究着重于星形胶质细胞在维持神经元功能中的作用，并尝试在转基因小鼠中引入Adiphtheia毒素A片段(DT-A)基因的细胞消融策略1)在建立转基因小鼠之前，从小鼠胚胎制备的原代培养细胞中检测星形胶质细胞特异性基因的表达。在这个项目的过程中，我们从神经生长锥富集部位纯化了几个与突触形成有关的蛋白质，测定了氨基酸序列，制备了针对这些多肽的特异性抗体，并分析了这些多肽在培养细胞中的亚细胞定位。2)基于时空特异性基因的表达，在星形胶质细胞中使用适当的启动子，如S100或GFAP启动子，DT-A基因在中枢神经系统的限制性细胞中被激活。此外，我们还使用了cre-lox系统来控制目的基因的表达。Cre重组酶介导的定点重组发生在34bp(LoxP)的限制性DNA序列上。小鼠Molony白血病病毒启动子下游插入转录终止信号可抑制DT-A活性。通过与cre重组酶共转染激活的dt-A导致细胞死亡。3)基因捕获提供了在中枢神经系统表达的新启动子。我们已经发现了与智力低下相关的陷阱基因，并表征了该基因表达对维持神经元功能的影响。

We focused on the role of astrocytes in maintaining of neuronal functions and attempted to introduce the strategy of cell ablation using a adiphtheia toxin A fragment (DT-A) gene in transgenic mice.1) Prior to create transgenic mice astrocyte specific gene expression was examined in primary culture cells prepared from mouse embryos. In the course of this project we purified several proteins involved in synapse formation from never growth cone-enriched fraction, determined the amino acid sequence, prepared specific antibodies against these peptides and analyzed subcellular localization in the cultured cells.2) Based on spatial-temporal specific gene expression using as appropriate promoter such as S100 or GFAP promoter in astrocyte, the DT-A gene is activated in the restricted cells in the central nervous system. In addition we used cre-lox system for control of the target gene expression. Cre-recombinase mediated site specific recombination occurs on the restricted DNA sequence composed of 34 bp (loxP). DT-A activity was suppressed by insertion of transcription termination signal down stream of murine molony leukemia virus promoter. The DT-A activated by co-transfection with cre-recombinase resulted in cell death.3) Gene trapping provided novel promoters expressed in the central nervous system. We have found the trapped gene related to mental retardation and characterize an affect of the gene expression on maintaining neuronal functions.