Main 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.

Contents:

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
Daphne 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,
OEzcan 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, OEzlem Tureci, and Ugur Sahin

12. Transfecting Human Monocytes with
RNA
Jens Dannull and Smita K. Nair

13.< In Vitro Synthesis, Delivery and
Bioavailability of Exogenous mRNA in Gene Transfer Mediated by PiggyBac Transposition
Solenne Bire, Nicole Ishac, and Florence
Rouleux-Bonnin

14. Transfection of Human Keratinocytes
with Nucleoside-Modified mRNA Encoding CPD-Photolyase to Repair DNA Damage
Gabor Boros, Katalin Kariko, Hiromi
Muramatsu, Edit Miko, Eszter Emri, Csaba Hegedus, Gabriella Emri, and Eva
Remenyik

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 OEzcan
Met

17. Transfection of Tumor-Infiltrating
T Cells with mRNA Encoding CXCR2
Manja Idorn, Per thor Straten, Inge Marie
Svane, and OEzcan 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 Frederic 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 Kariko

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