Published: June 10th, 2022
The protocol presents in vitro transcription (IVT) of chemically modified mRNA, cationic liposome preparation, and functional analysis of liposome enabled mRNA transfections in mammalian cells.
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.
As a therapeutic molecule, mRNA offers several advantages due to its non-integrative nature and its ability to transfect non-mitotic cells when compared to plasmid DNA (pDNA)1. Although mRNA delivery was demonstrated in the early 1990s, therapeutic applications were limited due to its lack of stability, its lack of immune activation, and poor translational efficiency2. Recently identified chemical modifications, such as pseudouridine 5'-triphosphate (Ψ-UTP) and methyl pseudouridine 5'-triphosphate (me1Ψ-UTP) on mRNA, helped to overcome these limitations, revolutionized mRNA research, and in turn, made m....
1. Production of me1 Ψ-UTP modified mRNA
We optimized the protocol for me1Ψ-UTP modified mRNA production, liposome preparation, and mRNA transfection experiments with cationic liposomes into multiple mammalian cells (Figure 1). To synthesize mRNA, the mammalian codon-optimized eGFP IVT template was amplified from the mEGFP-N1 mammalian expression vector and purified by organic extraction/ethanol precipitation method (Figure 2). Later, me1Ψ-UTP modified RNA and mRNA were produced by the IVT pr.......
Therapeutic applications of unmodified mRNAs have been limited due to their shorter half-life and their ability to activate intracellular innate immune responses, which in turn lead to poor protein expression in transfected cells11. Katalin et al. demonstrated that RNA containing modified nucleosides such as m5C, m6A, ΨU, and me1Ψ-UTP could avoid TLR activation12. More importantly, incorporation of ΨU or me1Ψ-UTP in IVT mRNA showed superior translational.......
MS thanks the Department of Biotechnology, India, for the financial support (BT/PR25841/GET/119/162/2017), Dr Alok Srivastava, Head, CSCR, Vellore, for his support and Dr Sandhya, CSCR core facilities for imaging and FACS experiments. We thank R. Harikrishna Reddy and Rajkumar Banerjee, Applied Biology Division, CSIR-Indian Institute of Chemical Technology Uppal Road, Tarnaka, Hyderabad, 500 007, TS, India, for their help in analyzing physico-chemical data of the liposomes. Vigneshwaran V, and Joshua A, CSCR for their help in video making.....
|Synthesized in the lab
|EDTA sodium salt
|Fetal bovine serum
|Gel documentation system
|Glacial acetic acid
|mEGFP-N1, Mammalian expression vector
|Phenol:chloroform:isoamyl alchol (25:24:1), pH 8.0
|Phosphate Buffer Saline (PBS), pH 7.4
|Sonics Vibra Cells
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