IDENTITY OF ALCOHOL ALTERED GENE EXPRESSION BY DDRT-PCR

描述：这项探索性/发展性赠款的目标是， 研究利用mRNA差异显示技术（DDRT-PCR）分离， 鉴定和开发乙醇诱导基因的分子探针 改变的表达用于未来对酒精中毒和相关疾病的研究 紊乱 这是了解后果的长期目标的一部分 慢性酒精诱导的大脑基因产物改变， 这有助于我们进一步了解酒精中毒的病因， 酒精相关性脑功能障碍的发病机制。 具体目标是 分离并测序在以下条件下差异表达基因转录物 慢性乙醇暴露的大鼠C6胶质瘤细胞的活化 脂多糖（LPS）+佛波醇酯（PMA）。 鉴定 在这些条件下差异表达的基因可以阐明 细胞内机制导致的适应不良过程 酒精引起的脑损伤，并提供洞察其他方面的 以未知的方式酗酒。 实验设计是执行 并排比较四组中的mRNA表达：1）未刺激的 对照细胞; 2）未刺激的慢性乙醇细胞; 3）激活对照 细胞; 4）激活的慢性乙醇细胞。 在隔离和验证后 激活的慢性乙醇细胞差异表达的mRNA， 将在活化的慢性乙醇细胞之间进行另外的比较 和激活的慢性乙醇戒断细胞。 激活条件 慢性治疗将用于证明乙醇抑制 诱导型一氧化氮合酶-2（NOS-2）在C6胶质瘤中的表达， 正常大鼠胶质细胞。 C6细胞在50 mM乙醇中生长9天 在暴露于LPS+PMA 24 h之前。 测定培养基亚硝酸盐以确认 慢性乙醇抑制NOS-2的表达，并提取总RNA， 细胞。 无DNA的RNA被逆转录成cDNA并通过PCR扩增。 使用针对差异显示优化的引物和条件的PCR 法 通过电泳分离PCR产物后， 通过放射自显影进行可视化，显示差异的cDNA条带 从凝胶上切下表达，通过PCR再扩增，并用于 北方印迹、克隆和测序。 基因与蛋白质杂交 序列数据库可以鉴定与已知基因和蛋白质的同源性;缺乏 同源性可以表示一个新的基因。

DESCRIPTION: The objective of this exploratory/developmental grant is, to investigate the use of differential display of mRNA (DDRT-PCR) to isolate, identify, and develop molecular probes for genes with ethanol-induced altered expression for use in future studies on alcoholism and associated disorders. This is part of a long-term goal to understand the consequences of chronic alcohol-induced alterations in brain gene products, knowledge of which can further our understanding of the etiology of alcoholism and the pathogenesis of alcohol-related brain disorders. The specific aim is to isolate and sequence gene transcripts differentially expressed following activation of chronic ethanol-exposed rat C6 glioma cells with lipopolysaccharide (LPS) + phorbol ester (PMA). Identification of differentially expressed genes under these conditions can elucidate intracellular mechanisms contributing to maladaptive processes underlying alcohol-induced brain damage, and provide insights into other aspects of alcoholism in as yet unknown ways. The experimental design is to perform side by-side comparisons of mRNA expression in four groups: 1) unstimulated control cells; 2) unstimulated chronic ethanol cells; 3) activated control cells; 4) activated chronic ethanol cells. Upon isolation and verification of mRNAs differentially expressed by activated chronic ethanol cells, an additional comparison will be made between activated chronic ethanol cells and activated chronic ethanol-withdrawn cells. Conditions for activation and chronic treatment will be those used to demonstrate ethanol-suppression of induced nitric oxide synthase-2 (NOS-2) expression in C6 glioma and normal rat glial cells. C6 cells are grown in 50 mM ethanol for 9 days prior to 24 h exposure to LPS+PMA. Media nitrite is assayed to confirm chronic ethanol suppressed NOS-2 expression, and total RNA is extracted from the cells. DNA-free RNA is reversed transcribed into cDNA and amplified by PCR using primers and conditions optimized for the differential display technique. Following separation of PCR products by electrophoresis and visualization by autoradiography, cDNA bands exhibiting differential expression are excised from the gel, re-amplified by PCR and used for Northern blotting, cloning and sequencing. Searches of gene and protein sequence databases may identify homology to known genes and proteins; lack of homology could denote a novel gene.