INHIBITION OF GENE EXPRESSION BY NORMAL & MODIFIED ANTISENSE OLIGONUCLEOTIDES

正常情况下基因表达的抑制

• 批准号: 3792518
• 负责人: C J MARCUS-SEKURA
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3792518
• 关键词: Epstein Barr virus; antisense nucleic acid; antiviral agents; chloramphenicol acetyltransferase; enzyme activity; enzyme linked immunosorbent assay; gene expression; genetic regulation; high performance liquid chromatography; immunofluorescence technique; monoclonal antibody; nucleic acid chemical synthesis; nucleic acid sequence; oligonucleotides; phorbols; protein purification; tissue /cell culture; transfection; virus antigen

This project was initially undertaken in order to determine whether modified oligonucleotides of appropriate antisense sequence can inhibit biological reactions, and to examine their potential as antiviral agents. A transient expression system in eukaryotic cells was used to see whether normal and modified oligonucleotides could inhibit the activity of the enzyme chloramphenicol transferase CAT (the gene for CAT was introduced into the cells by DNA transfection). It was determined that micromolar concentrations of modified oligonucleotides could inhibit CAT activity and that chain length, concentration, and nature of the chemical modification of the oligonucleotide could influence the degree of inhibition. This work has been published (1987) as well as a second paper discussing techniques for using antisense oligonucleotides has been published (1988). Experiments using antisense oligonucleotides have now been extended to study potential antiviral effects using EBV gene expression as a model. Antisense molecules appear to inhibit expression of a specific EBV early antigen, EA(D), induced by stimulation of B95-8 cells with the phorbol ester TPA as assayed by immunofluorescence and ELISA using a monoclonal antibody to EA(D). Additional antisense sequences are being examined to determine the specificity of this inhibition. We have purchased an instrument to synthesize oligonucleotides for these experiments and J. Smith has recently joined the laboratory, which should help greatly to supply materials which have been a limiting factor in this project. We have developed protocols for large-scale synthesis of antisense oligonucleotides and purification by HPLC, and implementation is planned in the near future. Temporarily, active research in this area has been curtailed due to time and personnel constraints.

该项目最初是为了确定是否 适当反义序列的修饰寡核苷酸可以抑制 生物反应，并检查其作为抗病毒药物的潜力 剂. 真核细胞中的瞬时表达系统用于 看看正常和修饰的寡核苷酸是否能抑制 氯霉素转移酶CAT（CAT的基因）的活性 通过DNA转染引入细胞）。 认定 微摩尔浓度的修饰寡核苷酸可以 抑制CAT活性和链长、浓度和性质 寡核苷酸的化学修饰可以影响 抑制程度。 这部作品已经出版（1987年）， 第二篇论文讨论了使用反义寡核苷酸的技术 已出版（1988）。 使用反义寡核苷酸的实验 现在已经扩展到使用EBV研究潜在的抗病毒作用 基因表达作为一个模型。 反义分子似乎抑制了 特异性EBV早期抗原EA（D）的表达， 用佛波醇酯TPA刺激B 95 -8细胞， 免疫荧光和ELISA使用EA的单克隆抗体（D）。 另外的反义序列正在检查中，以确定 这种抑制的特异性。 我们购买了一种仪器， 为这些实验合成寡核苷酸，J. Smith已经 最近加入了实验室，这将大大有助于供应 这些材料是这个项目的一个限制因素。 我们有 开发了大规模合成反义核酸的方案， 寡核苷酸和HPLC纯化，并计划实施 在近期 目前，这一领域的积极研究已经 由于时间和人员的限制，