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（改编自研究者摘要）：一种常规的