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: 2900929
• 负责人: MOO J CHO
• 金额: $ 10.1万
• 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 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2900929
• 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（改编自研究者摘要）：常规 用于评估反义核酸的细胞递送的功能测定系统 药物通常是基于基因翻译步骤的下调 通过反义试剂的表达。 该程序受到几个 理论和实际问题。 在拟议的研究中，PI 计划设计一种细胞分析系统， 以一种新的蛋白质， 荧光素酶在本例中，开始作为反义试剂出现， 进入细胞。 该系统可以通过用一种 具有异常剪接的荧光素酶质粒，其可以通过 反义试剂。 将通过以下方法制备至少三种不同的重组荧光素酶质粒： 将突变的人b-珠蛋白内含子2插入到 来自商业来源的荧光素酶质粒pTRE-LUC。 最近，PI 表明突变内含子引起的异常剪接可以 通过靶向其5 ′剪接位点的17-mer反义进行校正。 中 初步实验中，PI发现转染的HeLa细胞 当寡聚体 通过Lipofectamine引入细胞，清楚地建立了 可行性 除HeLa细胞外，成纤维细胞系NIH/3 T3和 单核细胞衍生的Raw 264.7细胞将用质粒转染。 这些细胞将表现出不同的内吞活性。 当 发展，目前的系统将是最有用的，并可能成为一个 通用分析系统，客观地比较各种新的战略， 反义/抗原递送。 然而，由于其内在的性质， 系统不能与激活RNase H的寡聚体一起使用。但这 不是限制。