ASSAY SYSTEM FOR EVALUATING CELLULAR ANTISENSE DELIVERY

用于评估细胞反义递送的测定系统

• 批准号: 2602743
• 负责人: MOO J CHO
• 金额: $ 9.63万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2602743
• 关键词: 3T3 cells; HeLa cells; RNA splicing; antigen presentation; antigen presenting cell; antisense nucleic acid; bioassay; biotechnology; drug delivery systems; enzyme biosynthesis; luciferin monooxygenase; oligonucleotides; plasmids; protein biosynthesis; recombinant DNA; transfection

DESCRIPTION (Adapted from Investigator's Abstract): A conventional functional assay system for assessing cellular delivery of an antisense agent is usually based on down-regulation of the translational step in gene expression by the antisense agent. The procedure suffers from several theoretical as well as practical problems. In the proposed study, the PI plans to devise a cellular assay system in which an antisense agent up-regulates the protein synthesis in such a way that a new protein, luciferase in the present case, begins to appear as the antisense agent enters the cell. The system can be developed by transfecting cells with a luciferase plasmid with aberrant splicing that can be corrected by an antisense agent. At least three different recombinant luciferase plasmids will be prepared by inserting a mutant human b-globin intron 2 at different locations of a luciferase plasmid pTRE-LUC from a commercial source. Recently, the PI has shown that the aberrant splicing caused by the mutant intron can be corrected by a 17-mer antisense targeted to its 5' splice site. In a preliminary experiment, the PI found that the HeLa cells transfected by the recombinant luciferase plasmid indeed produce luciferase when the oligomer was introduced to the cell by Lipofectamine, clearly establishing feasibility. In addition to HeLa cells, fibroblast cell line NIH/3T3 and monocyte-derived Raw 264.7 cells will be transfected with the plasmid. These cells will demonstrate different endocytic activities. When developed, the present system will be most useful and could become a universal assay system in objectively comparing various novel strategies of antisense/antigen delivery. Due to its intrinsic nature, however, ther system cannot be used with an oligomer that activates RNase H. However, this is not a limitation.

描述（根据调查员的摘要改编）：一种传统的 用于评估反义细胞递送的功能测定系统 代理通常基于基因翻译步骤的下调 反义剂的表达。 该过程有几个 理论和实际问题。 在拟议的研究中，PI 计划设计一种反义剂的蜂窝测定系统 上调蛋白质合成的方式使得新蛋白质， 在本案中，荧光素酶开始出现为反义剂 进入细胞。 该系统可以通过用A转染细胞来开发 荧光素酶质粒具有异常剪接，可以通过 反义剂。 至少三种不同的重组荧光素酶质粒将由 在A的不同位置插入突变的人B-珠蛋白内含子2 来自商业来源的荧光素酶质粒ptre-luc。 最近，Pi有 表明突变内含子引起的异常剪接可以是 通过针对其5'剪接部位的17-Mer反义纠正。 在 初步实验，PI发现HELA细胞由 重组荧光素酶质粒确实会产生荧光素酶 是由脂肪欣胺引入细胞的，明确建立了 可行性。 除HeLa细胞外，成纤维细胞系NIH/3T3和 单核细胞衍生的264.7细胞将用质粒转染。 这些细胞将表现出不同的内吞作用。 什么时候 开发，当前系统将是最有用的，并且可能成为 通用测定系统在客观地比较各种新颖策略 反义/抗原递送。 然而，由于其内在性质 系统不能与激活RNase H的低聚物一起使用。但是，此 不是限制。