Centre for Stem Cell Research, Christian Medical College Campus.
Centre for Stem Cell Research, Christian Medical College Campus;
J Vis Exp. 2022 Jun 10(184). doi: 10.3791/62407.
In recent years, chemically modified messenger RNA (mRNA) has emerged as a potent nucleic acid molecule for developing a wide range of therapeutic applications, including a novel class of vaccines, protein replacement therapies, and immune therapies. Among delivery vectors, lipid nanoparticles are found to be safer and more effective in delivering RNA molecules (e.g., siRNA, miRNA, mRNA) and a few products are already in clinical use. To demonstrate lipid nanoparticle-mediated mRNA delivery, we present an optimized protocol for the synthesis of functional me1Ψ-UTP modified eGFP mRNA, the preparation of cationic liposomes, the electrostatic complex formation of mRNA with cationic liposomes, and the evaluation of transfection efficiencies in mammalian cells. The results demonstrate that these modifications efficiently improved the stability of mRNA when delivered with cationic liposomes and increased the eGFP mRNA translation efficiency and stability in mammalian cells. This protocol can be used to synthesize the desired mRNA and transfect with cationic liposomes for target gene expression in mammalian cells.
近年来，化学修饰的信使 RNA（mRNA）作为一种强大的核酸分子，已经在广泛的治疗应用中崭露头角，包括一类新型疫苗、蛋白质替代疗法和免疫疗法。在递药载体中，脂质纳米粒在递送 RNA 分子（如 siRNA、miRNA、mRNA）方面被发现更安全、更有效，并且已经有一些产品在临床应用中。为了证明脂质纳米粒介导的 mRNA 递呈，我们提出了一种优化的方案，用于合成功能性 me1Ψ-UTP 修饰的 eGFP mRNA、阳离子脂质体的制备、mRNA 与阳离子脂质体的静电复合物形成以及在哺乳动物细胞中转染效率的评估。结果表明，这些修饰在与阳离子脂质体共递呈时能有效地提高 mRNA 的稳定性，并增加 eGFP mRNA 在哺乳动物细胞中的翻译效率和稳定性。该方案可用于合成所需的 mRNA，并与阳离子脂质体共转染以在哺乳动物细胞中表达靶基因。
