INHIBITION OF GENE EXPRESSION BY NORMAL & MODIFIED ANTISENSE OLIGONUCLEOTIDES

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，并检测它们作为抗病毒的潜力 探员们。利用真核细胞中的瞬时表达系统 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基因)的活性 通过DNA转染法导入细胞)。它被确定为 修饰的寡核苷酸的微摩尔浓度可以 抑制CAT活性以及其链长、浓度和性质 寡核苷酸的化学修饰可能会影响 抑制程度。这项工作已经出版(1987)以及一个 第二篇论文讨论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 现已扩展到使用EBV研究潜在的抗病毒作用 以基因表达为模型。反义分子似乎能抑制 重组人EB病毒早期抗原EA(D)的诱导表达 佛波酯TPA对B95-8细胞的刺激作用 用抗EA(D)的单抗进行免疫荧光和ELISA法。 正在检测更多的反义序列以确定 这种抑制的特异性。我们已经购买了一台仪器来 为这些实验合成寡核苷酸，J·史密斯 最近加入了实验室，这应该会对供应有很大帮助 材料一直是这个项目的一个限制因素。我们有 开发的大规模合成反义寡核苷酸的方案 寡核苷酸和高效液相纯化，并计划实施 在不久的将来。目前，这一领域的活跃研究一直是 由于时间和人员限制，裁员。

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.