INHIBITION OF GENE EXPRESSION BY NORMAL & MODIFIED ANTISENSE OLIGONUCLEOTIDES

正常情况下基因表达的抑制

• 批准号: 3804790
• 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/3804790
• 关键词: Epstein Barr virus; antisense nucleic acid; antiviral agents; chloramphenicol acetyltransferase; 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; 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 limitting factor in this project. We have developed protocols for largescale synthesis of antisense oligonucleotides and purification by HPLC, and implementation is planned in the near future.

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，并检查它们作为抗病毒剂的潜力。 使用真核细胞中的瞬时表达系统来观察是否 正常和修饰的寡核苷酸可以抑制 氯霉素转移酶CAT（引入CAT基因 通过DNA转染进入细胞）。 经测定，微摩尔 修饰寡核苷酸的浓度可以抑制 CAT 活性 化学修饰的链长、浓度和性质 寡核苷酸的变化可能影响抑制程度。这部作品 已出版（1987）以及第二篇讨论技术的论文 使用反义寡核苷酸的方法已发表（1988）。 使用反义寡核苷酸的实验现已扩展到 使用 EBV 基因表达作为模型研究潜在的抗病毒作用。 反义分子似乎可以早期抑制特定 EBV 的表达 抗原 EA (D)，通过用佛波醇刺激 B95-8 细胞诱导 使用单克隆抗体通过免疫荧光和 ELISA 测定酯 TPA EA(D) 抗体。 正在检查其他反义序列 确定这种抑制的特异性。 我们购买了一个 用于合成这些实验的寡核苷酸的仪器和 J. 史密斯最近加入了该实验室，这应该会对 供应材料一直是该项目的限制因素。 我们 开发了大规模合成反义的方案 寡核苷酸和 HPLC 纯化，并计划实施 在不久的将来。