IDENTITY OF ALCOHOL ALTERED GENE EXPRESSION BY DDRT-PCR

描述：这项探索性/发展性赠款的目的是 探讨应用差异显示技术(DDRT-PCR)分离沙门氏菌 鉴定和开发乙醇诱导基因的分子探针 改变表达以用于未来对酒精中毒和相关的研究 精神错乱。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的改变，知识 这可以加深我们对酒精中毒的病因和 酒精相关脑部疾病的发病机制。其具体目的是 分离和测序下列差异表达的基因转录本 乙醇对慢性暴露大鼠C6胶质瘤细胞的激活作用 脂多糖+佛波酯(PMA)。身份识别 在这些条件下差异表达的基因可以解释 导致不良适应过程的细胞内机制 酒精引起的脑损伤，并提供对以下方面的洞察 以一种未知的方式酗酒。实验设计是为了执行 四组基因表达的并列比较：1)未受刺激 对照细胞；2)未受刺激的慢性乙醇细胞；3)激活对照 细胞；4)激活慢性乙醇细胞。在隔离和验证之后 在激活的慢性乙醇细胞差异表达的mRNA中， 将在激活的慢性乙醇细胞之间进行额外的比较 并激活慢性酒精戒断细胞。激活条件 慢性治疗将被用来证明酒精抑制。 诱导型一氧化氮合酶-2在C6胶质瘤中的表达 正常大鼠神经胶质细胞。C6细胞在50 mM乙醇中培养9天 脂多糖+PMA染毒前24小时。对媒体亚硝酸盐进行检测以确认 慢性乙醇抑制一氧化氮合酶-2表达，总RNA从 这些细胞。不含DNA的RNA被逆转录成cDNA，并通过 用于差异显示的引物和条件的优化 技术。聚合酶链式反应产物经电泳法分离后 放射自显影显示差异条带 从凝胶中切下表达，通过聚合酶链式反应重新扩增，并用于 Northern杂交、克隆和测序。基因和蛋白质的搜索 序列数据库可能识别与已知基因和蛋白质的同源性；缺乏 同源性可能表示一种新的基因。

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cells, an additional comparison will be made between activated chronic ethanol cells and activated chronic ethanol-withdrawn cells.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.