Synthetic mRNA: Production, Introduction into Cells, and Physiological Consequences: 2016 (Methods in Molecular Biology)

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 Introduction 1. Synthetic mRNA: Production, Introduction into Cells, and Physiological Consequences Robert E. Rhoads Part II Synthesis of mRNA 2. Synthetic Capped mRNAs for Cap-Specific Photo-Crosslinking Experiments Joanna Kowalska, Franck Martin, and Jacek Jemielity 3. Enzymatic Modification of 5'-Capped RNA and Subsequent Labeling by Click Chemistry Josephin M. Holstein, Daniela Stummer, and Andrea Rentmeister 4. Preparation of Functional, Fluorescently Labeled mRNA Capped with Anthraniloyl-m7GpppG Artem V. Domashevskiy, David J. Rodriguez, Dilantha Gunawardana, and Dixie J. Goss 5. Intronless b-Globin Reporter: A Tool for Studying Nuclear RNA Stability Elements Jessica A. Brown and Joan A. Steitz 6. Synthetic mRNA with Superior Properties that Mimics the Intracellular Fates of Natural Histone mRNA Wei Su, Michael K. Slevin, William F. Marzluff, and Robert E. Rhoads 7. Engineering WT1-Encoding mRNA to Increase Translational Efficiency in Dendritic Cells Daphn Benteyn, Carlo Heirman, Kris Thielemans, and Aude Bonehill Part III Introduction of Synthetic mRNA into Cells 8. Electroporation of Alphavirus RNA Translational Reporters into Fibroblastic and Myeloid Cells as a Tool to Study the Innate Immune System Christina L. Gardner, Derek W. Trobaugh, Kate D. Ryman, and William B. Klimstra 9. GMP-Grade mRNA Electroporation of Dendritic Cells for Clinical Use Judith Derdelinckx, Zwi N. Berneman, and Nathalie Cools 10. Large Scale mRNA Transfection of Dendritic Cells by Electroporation in Continuous Flow Systems David Selmeczi, Thomas Steen Hansen, zcan Met, Inge Marie Svane, and Niels B. Larsen 11. FLT3 Ligand as a Molecular Adjuvant for Naked RNA Vaccines Sebastian Kreiter, Mustafa Diken, Abderraouf Selmi, Jutta Petschenka, zlem Treci, and Ugur Sahin 12. Transfecting Human Monocytes with RNA Jens Dannull and Smita K. Nair 13. Part IV Alteration of Cellular Function with Synthetic mRNA 15. Delivery of Synthetic mRNA Encoding FOXP3 Antigen into Dendritic Cells for Inflammatory Breast Cancer Immunotherapy Gayathri R. Devi and Sritama Nath 16. Immune Monitoring using mRNA-Transfected Dendritic Cells Troels Holz Borch, Inge Marie Svane, and zcan Met 17. Transfection of Tumor-Infiltrating T Cells with mRNA Encoding CXCR2 Manja Idorn, Per thor Straten, Inge Marie Svane, and zcan Met 18. mRNA Electroporation of Dendritic Cells with WT1, Survivin and TriMix (A Mixture of caTLR4, CD40L, and CD70) An Coosemans, Sandra Tuyaerts, Kim Morias, Jurgen Corthals, Carlo Heirman, Kris Thielemans, Stefaan W. Van Gool, Ignace Vergote, and Frdric Amant 19. Redirecting T Cell Specificity Using T Cell Receptor Messenger RNA Electroporation Sarene Koh, Noriko Shimasaki, and Antonio Bertoletti 20. Measuring Hematocrit in Mice Injected with In Vitro-Transcribed Erythropoietin mRNA Azita Josefine Mahiny and Katalin Karik 21. Genetic Modification of Human Pancreatic Progenitor Cells through Modified mRNA Song Lu, Christie C. Chow, Junwei Zhou, Po Sing Leung, Stephen K. Tsui, and Kathy O. Lui