<em>In Vitro</em> Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

Abstract

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express. Video LinkThe video component of this article can be found at

In VitroEvaluation of a Novel mRNA-Based Therapeutic Strategy for the Treatment of Patients Suffering from Alpha-1-Antitrypsin Deficiency

“…IVT was performed as previously described [32]. In brief, the pEX-K4 vector containing the coding sequence for human AAT (Eurofins Medigenomix GmbH, Ebersberg, Germany) was amplified using the HotStar HiFidelity Polymerase Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.…”

V-Maf Musculoaponeurotic Fibrosarcoma Oncogene Homolog A Synthetic Modified mRNA Drives Reprogramming of Human Pancreatic Duct-Derived Cells Into Insulin-Secreting Cells

“…In our system, efficient smRNA‐to‐protein translation was shown in HDDCs by detection of cytoplasmic fluorescence induced by EGFP smRNA and by nuclear localization of MAFA protein after MAFA smRNA transfection. According to protein expression levels, we estimate our transfection efficiency to be between 35% and 50% with EGFP and MAFA smRNAs, which is slightly lower than other studies reporting higher transfection rates (50%–100%) [23, 27, 37, 41, 42], although performed either in rodent adult or human fetal tissues. MAFA protein was able to bind to human insulin promoter in HEK293 cells that were used for their capacity to efficiently overexpress ectopic and recombinant proteins [43].…”

“…RNA‐based reprogramming is regarded as a strong alternative to DNA‐based techniques to generate defined cell types for clinical application [24, 36, 37]. In 2010, Warren et al demonstrated the successful smRNA‐mediated reprogramming of human fibroblast to induced pluripotent stem cells (iPSCs) in vitro [27] using transcripts for KLF4 , c‐MYC , OCT4, and SOX2 .…”

“…After VEGF‐A overexpression, histology showed reduced infarct size and concomitant expansion and differentiation of endogenous heart progenitors cells. For clinical perspectives, RNA‐based reprogramming bears the advantage over DNA‐based techniques to be devoid of risk for insertional mutagenesis and genomic integration that may disrupt cell cycle control and lead to outgrowth of transplanted cells [24, 36, 37].…”

Light‐Activated Control of Translation by Enzymatic Covalent mRNA Labeling

“…IVT-mod-mRNA encoding EGFP (mod-RNA-Tag1/2/3 and mod-RNA-Tag0) could be conveniently synthesized following a previously established method. [31] The IVT product was subsequently polyadenylated and labeled by TGT with compound 7 . HeLa cells were transiently transfected with the photocaged mod-mRNA and illuminated with a 405 nm laser as previously described.…”