荧光素酶mRNA的构建及电穿孔介导的mRNA体内表达特性

Preparation of luciferase-expressing mRNA and expression characteristics of mRNA delivered by electroporation in vivo

摘要:

为构建一种非复制型mRNA平台并探究电穿孔介导的mRNA对小鼠健康状况的影响及蛋白的表达情况，以荧光素酶作为靶标基因，用T7 RNA聚合酶体外转录及酶法加帽加尾的策略制备mRNA，用活体基因导入仪通过电穿孔的方式体内递送mRNA，借助小动物活体成像系统观测荧光素酶蛋白在小鼠体内的表达强度和持续时间。结果表明，使用该非复制型mRNA平台得到的mRNA成功在体内外表达，电穿孔介导的mRNA对小鼠健康体征无明显影响，所有的小鼠均成功表达了荧光素酶蛋白，蛋白表达在电穿孔后第1天达到峰值，在第4天迅速下降，但蛋白表达强度和持续时间存在较大的小鼠个体间差异。研究对非复制型mRNA的构建及其应用于疫苗或肿瘤药物研发具有重要参考价值。

Abstract:

In this study, we aimed to construct a non-replication mRNA platform and explore the side effects of electroporation-mediated delivery of mRNA on the mice as well as the expression features of the mRNA. With luciferase gene as a marker, in vitro transcription with T7 RNA polymerase was carried out for the synthesis of luciferase-expressed mRNA, followed by enzymatic capping and tailing. The mRNA was delivered in vivo by electroporation via an in vivo gene delivery system, and the expression intensity and duration of luciferase in mice were observed via an in vivo imaging system. The results demonstrated that the mRNA transcripts were successfully expressed both in vitro and in vivo. The electroporation-mediated delivery of mRNA had no obvious side effects on the mice. Luciferase was expressed successfully in all the mRNA-transduced mice, while the expression intensity and duration varied among individuals. Overall, the expression level peaked on the first day after electroporation and rapidly declined on the fourth day. This study is of great importance for the construction of non-replication mRNAs and their application in vaccine or antitumor drug development.