This volumepresents detailed laboratory protocols for invitro synthesis of mRNA with favorable properties, its introduction intocells by a variety of techniques, and the measurement of physiological andclinical consequences such as protein replacement and cancer immunotherapy. Synthetictechniques are described for structural features in mRNA that provide investigationaltools such as fluorescence emission, click chemistry, photo-chemicalcrosslinking, and that produce mRNA with increased stability in the cell, increased translational efficiency, and reducedactivation of the innate immune response. Protocols are described for clinicalapplications such as large-scale transfection of dendritic cells, production ofGMP-grade mRNA, redirecting T cell specificity, and use of molecularadjuvants for RNA vaccines. Writtenin the highly successful Methods inMolecular Biology series format, chapters include introductions to theirrespective topics, lists of the necessary materials and reagents, step-by-steplaboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

SyntheticmRNA: Production, Introduction into Cells, and Physiological Consequences is a valuable and cutting-edge resourcefor both laboratory investigators and clinicians interested in this powerfuland rapidly evolving technology.

Part I Introduction

1. Synthetic mRNA: Production,Introduction into Cells, and Physiological Consequences
Robert E. Rhoads

PartII Synthesis of mRNA

2.     SyntheticCapped mRNAs for Cap-Specific Photo-Crosslinking Experiments
Joanna Kowalska, Franck Martin, and Jacek Jemielity

3.      Enzymatic Modification of 5´-CappedRNA and Subsequent Labeling by Click Chemistry
Josephin M. Holstein, Daniela Stummer, andAndrea 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-GlobinReporter: A Tool for Studying NuclearRNA Stability Elements
Jessica A. Brown and Joan A. Steitz

6.     Synthetic mRNA with Superior Properties that Mimicsthe Intracellular Fates of Natural Histone mRNA
Wei Su, Michael K. Slevin,William F. Marzluff, and Robert E.Rhoads

7.      Engineering WT1-Encoding mRNA toIncrease Translational Efficiency in Dendritic Cells
Daphné Benteyn, Carlo Heirman, KrisThielemans, and Aude Bonehill

PartIII Introduction of Synthetic mRNA into Cells

8. Electroporation of Alphavirus RNATranslational Reporters into Fibroblastic and Myeloid Cells as a Tool to Studythe Innate Immune System
Christina L. Gardner, Derek W. Trobaugh,Kate D. Ryman, and William B. Klimstra

9. GMP-Grade mRNA Electroporation ofDendritic Cells for Clinical Use
Judith Derdelinckx, Zwi N. Berneman, and NathalieCools

10. Large Scale mRNA Transfection ofDendritic Cells by Electroporation in Continuous Flow Systems
David Selmeczi, Thomas Steen Hansen,Özcan Met, Inge Marie Svane, and Niels B. Larsen

11. FLT3 Ligand as a Molecular Adjuvantfor Naked RNA Vaccines
Sebastian Kreiter, Mustafa Diken,Abderraouf Selmi, Jutta Petschenka, Özlem Türeci, and Ugur Sahin

12. Transfecting Human Monocytes withRNA
Jens Dannull and Smita K. Nair

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

14. Transfection of Human Keratinocyteswith Nucleoside-Modified mRNA Encoding CPD-Photolyase to Repair DNA Damage
Gábor Boros, Katalin Karikó, HiromiMuramatsu, Edit Miko, Eszter Emri, Csaba Hegedus, Gabriella Emri, and ÉvaRemenyik

PartIV Alteration of Cellular Function with Synthetic mRNA

15. Delivery of Synthetic mRNA EncodingFOXP3 Antigen into Dendritic Cells for Inflammatory Breast Cancer Immunotherapy

Gayathri R. Devi and Sritama Nath
16. Immune Monitoring using mRNA-TransfectedDendritic Cells
Troels Holz Borch, Inge Marie Svane, and ÖzcanMet

17. Transfection of Tumor-InfiltratingT Cells with mRNA Encoding CXCR2
Manja Idorn, Per thor Straten, Inge MarieSvane, and Özcan Met

18. mRNA Electroporation of DendriticCells with WT1, Survivin and TriMix (A Mixture of caTLR4, CD40L, and CD70)
An Coosemans, Sandra Tuyaerts, KimMorias, Jurgen Corthals, Carlo Heirman, Kris Thielemans, Stefaan W. Van Gool,Ignace Vergote, and Frédéric Amant

19. Redirecting T Cell SpecificityUsing T Cell Receptor Messenger RNA Electroporation
Sarene Koh, Noriko Shimasaki, and AntonioBertoletti

20. Measuring Hematocrit in Mice Injectedwith In Vitro-Transcribed Erythropoietin mRNA
Azita Josefine Mahiny and Katalin Karikó

21. Genetic Modification of HumanPancreatic Progenitor Cells through Modified mRNA
Song Lu, Christie C. Chow, Junwei Zhou,Po Sing Leung, Stephen K. Tsui, and Kathy O. Lui

This volumepresents detailed laboratory protocols for invitro synthesis of mRNA with favorable properties, its introduction intocells by a variety of techniques, and the measurement of physiological andclinical consequences such as protein replacement and cancer immunotherapy. Synthetictechniques are described for structural features in mRNA that provide investigationaltools such as fluorescence emission, click chemistry, photo-chemicalcrosslinking, and that produce mRNA with increased stability in the cell, increased translational efficiency, and reducedactivation of the innate immune response. Protocols are described for clinicalapplications such as large-scale transfection of dendritic cells, production ofGMP-grade mRNA, redirecting T cell specificity, and use of molecularadjuvants for RNA vaccines. Writtenin the highly successful Methods inMolecular Biology series format, chapters include introductions to theirrespective topics, lists of the necessarymaterials and reagents, step-by-steplaboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

SyntheticmRNA: Production, Introduction into Cells, and Physiological Consequences is a valuable and cutting-edge resourcefor both laboratory investigators and clinicians interested in this powerfuland rapidly evolving technology.

Synthetic mRNA: Production, Introduction into Cells, and Physiological Consequences.- Synthetic Capped mRNAs for Cap-Specific Photo-Crosslinking Experiments.- Enzymatic Modification of 5'-Capped RNA and Subsequent Labeling by Click Chemistry.- Preparation of Functional, Fluorescently Labeled mRNA Capped with Anthraniloyl-m⁷GpppG.- Intronless b-Globin Reporter: A Tool for Studying Nuclear RNA Stability Elements.- Synthetic mRNA with Superior Properties that Mimics the Intracellular Fates of Natural Histone mRNA.- Engineering WT1-Encoding mRNA to Increase Translational Efficiency in Dendritic Cells.- Electroporation of Alphavirus RNA Translational Reporters into Fibroblastic and Myeloid Cells as a Tool to Study the Innate Immune System.- GMP-Grade mRNA Electroporation of Dendritic Cells for Clinical Use.- Large Scale mRNA Transfection of Dendritic Cells by Electroporation in Continuous Flow Systems.- FLT3 Ligand as a Molecular Adjuvant for Naked RNA Vaccines.- Transfecting Human Monocytes with RNA.- In Vitro Synthesis, Delivery and Bioavailability of Exogenous mRNA in Gene Transfer Mediated byPiggyBac Transposition.- Transfection of Human Keratinocytes with Nucleoside-Modified mRNA Encoding CPD-Photolyase to Repair DNA Damage.- Delivery of Synthetic mRNA Encoding FOXP3 Antigen into Dendritic Cells for Inflammatory Breast Cancer Immunotherapy.- Immune Monitoring using mRNA-Transfected Dendritic Cells.- Transfection of Tumor-Infiltrating T Cells with mRNA Encoding CXCR2.- mRNA Electroporation of Dendritic Cells with WT1, Survivin and TriMix (A Mixture of caTLR4, CD40L, and CD70).- Redirecting T Cell Specificity Using T Cell Receptor Messenger RNA Electroporation.- Measuring Hematocrit in Mice Injected with In Vitro-Transcribed Erythropoietin mRNA.- Genetic Modification of Human Pancreatic Progenitor Cells through Modified mRNA.

Inhaltsverzeichnis

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
Daphné 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, Özcan 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, Özlem Türeci, 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
Gábor Boros, Katalin Karikó, Hiromi Muramatsu, Edit Miko, Eszter Emri, Csaba Heged¿s, Gabriella Emri, and Éva 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 Özcan Met

17. Transfection of Tumor-Infiltrating T Cells with mRNA Encoding CXCR2
Manja Idorn, Per thor Straten, Inge Marie Svane, and Özcan 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 Frédéric 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 Karikó

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
Klappentext

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 necessarymaterials 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.

Includes cutting-edge methods and protocols
Provides step-by-step detail essential for reproducible results
Contains key notes and implementation advice from the experts
Includes supplementary material: sn.pub/extras

Synthetic mRNA (Englisch)Production, Introduction Into Cells, and Physiological ConsequencesRhoads, Robert E.

140,45 €

Synthetic mRNA

Produktbeschreibung

Inhaltsverzeichnis

Klappentext

leseprobe

Synthetic mRNA
(Englisch)
Production, Introduction Into Cells, and Physiological Consequences
Rhoads, Robert E.