The role of astrocytes in maintaing reuronal function

我们关注星形胶质细胞在维持神经元功能中的作用，并试图在转基因小鼠中引入使用adiphtheia毒素a片段（DT-A）基因的细胞消融策略。1)在创建转基因小鼠星形胶质细胞之前，在小鼠胚胎制备的原代培养细胞中检测了特异性基因表达。在这个项目的过程中，我们从从未生长锥体富集的部分中纯化了几个参与突触形成的蛋白质，确定了氨基酸序列，制备了针对这些肽的特异性抗体，并分析了培养细胞的亚细胞定位。2)利用星形胶质细胞中合适的启动子如S100或GFAP启动子，基于时空特异性基因表达，在中枢神经系统的限制性细胞中激活DT-A基因。此外，我们使用cre-lox系统来控制目标基因的表达。cre -重组酶介导的位点特异性重组发生在由34 bp （loxP）组成的限制性DNA序列上。在小鼠白血病启动子下游插入转录终止信号抑制了DT-A的活性。与cree -recombinase共转染激活的DT-A导致细胞死亡。3)基因诱捕提供了在中枢神经系统表达的新启动子。我们发现了与智力迟钝相关的被困基因，并描述了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.