Description
This volume presents detailed laboratory protocols for in vitro synthesis of mRNA with favorable properties, its introduction into cells by a variety of techniques, and the measurement of physiological and clinical consequences such as protein replacement and cancer immunotherapy. Synthetic techniques are described for structural features in mRNA that provide investigational tools such as fluorescence emission, click chemistry, photo-chemical crosslinking, and that produce mRNA with increased stability in the cell, increased translational efficiency, and reduced activation of the innate immune response. Protocols are described for clinical applications such as large-scale transfection of dendritic cells, production of GMP-grade mRNA, redirecting T cell specificity, and use of molecular adjuvants for RNA vaccines. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Synthetic mRNA: Production, Introduction into Cells, and Physiological Consequences is a valuable and cutting-edge resource for both laboratory investigators and clinicians interested in this powerful and rapidly evolving technology. Part I Introduction1. Synthetic mRNA: Production, Introduction into Cells, and Physiological ConsequencesRobert E. Rhoads Part II Synthesis of mRNA2. Synthetic Capped mRNAs for Cap-Specific Photo-Crosslinking ExperimentsJoanna Kowalska, Franck Martin, and Jacek Jemielity3. Enzymatic Modification of 5′-Capped RNA and Subsequent Labeling by Click ChemistryJosephin M. Holstein, Daniela Stummer, and Andrea Rentmeister4. Preparation of Functional, Fluorescently Labeled mRNA Capped with Anthraniloyl-m7GpppGArtem V. Domashevskiy, David J. Rodriguez, Dilantha Gunawardana, and Dixie J. Goss5. Intronless b-Globin Reporter: A Tool for Studying Nuclear RNA Stability ElementsJessica A. Brown and Joan A. Steitz6. Synthetic mRNA with Superior Properties that Mimics the Intracellular Fates of Natural Histone mRNAWei Su, Michael K. Slevin, William F. Marzluff, and Robert E. Rhoads7. Engineering WT1-Encoding mRNA to Increase Translational Efficiency in Dendritic CellsDaphn Benteyn, Carlo Heirman, Kris Thielemans, and Aude BonehillPart III Introduction of Synthetic mRNA into Cells8. Electroporation of Alphavirus RNA Translational Reporters into Fibroblastic and Myeloid Cells as a Tool to Study the Innate Immune SystemChristina L. Gardner, Derek W. Trobaugh, Kate D. Ryman, and William B. Klimstra9. GMP-Grade mRNA Electroporation of Dendritic Cells for Clinical UseJudith Derdelinckx, Zwi N. Berneman, and Nathalie Cools10. Large Scale mRNA Transfection of Dendritic Cells by Electroporation in Continuous Flow SystemsDavid Selmeczi, Thomas Steen Hansen, zcan Met, Inge Marie Svane, and Niels B. Larsen11. FLT3 Ligand as a Molecular Adjuvant for Naked RNA VaccinesSebastian Kreiter, Mustafa Diken, Abderraouf Selmi, Jutta Petschenka, zlem Treci, and Ugur Sahin12. Transfecting Human Monocytes with RNAJens Dannull and Smita K. Nair13.
