title: In vitro 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.
authors.affiliation: Department of Thoracic, Cardiac, and Vascular Surgery, University Hospital Tuebingen; meltem.avci-adali@klinikum.uni-tuebingen.de.
authors.forename: Meltem
authors.initials: M
authors.lastname: Avci-Adali
authors.affiliation: Department of Thoracic, Cardiac, and Vascular Surgery, University Hospital Tuebingen.
authors.forename: Andreas
authors.initials: A
authors.lastname: Behring
authors.affiliation: Department of Thoracic, Cardiac, and Vascular Surgery, University Hospital Tuebingen.
authors.forename: Heidrun
authors.initials: H
authors.lastname: Steinle
authors.affiliation: Department of Thoracic, Cardiac, and Vascular Surgery, University Hospital Tuebingen.
authors.forename: Timea
authors.initials: T
authors.lastname: Keller
authors.affiliation: Department of Thoracic, Cardiac, and Vascular Surgery, University Hospital Tuebingen.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Krajeweski
authors.affiliation: Department of Thoracic, Cardiac, and Vascular Surgery, University Hospital Tuebingen.
authors.forename: Christian
authors.initials: C
authors.lastname: Schlensak
authors.affiliation: Department of Thoracic, Cardiac, and Vascular Surgery, University Hospital Tuebingen.
authors.forename: Hans P
authors.initials: HP
authors.lastname: Wendel
mesh_terms: D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D010957:Plasmids / Q000008:administration & dosage / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000008:administration & dosage* / Q000138:chemical synthesis* / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D012333:RNA, Messenger; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins