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“Next generation” sequencing techniques allow for more detailed analysis of exons and introns in multiple genes at the same time. This will reveal many mutations that potentially lead to exon skipping. To functionally test these a lot can be achieved with a limited set of protocols, while for the intentional induction of exon skipping different tools and target genes are involved and the translational path from in vitro splicing to in vivo tests in animal models requiring a more extensive set of protocols. Exon Skipping: Methods and Protocols provides scientist with a comprehensive guide to many of the methods and techniques used for exon skipping, such as methods on how to discriminate “real polymorphisms” from mutations that affect splicing. 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, readily reproducible laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical Exon Skipping: Methods and Protocols seeks to aid scientists in the continuing study of exon skipping.
Part I: Mutations
1. DNA Diagnostics and Exon Skipping
Umasuthan Srirangalingam and Shern L. Chew
2. Bioinformatics and Mutations Leading to Exon Skipping
F.O. Desmet and C. Béroud
3. Minigenes to Confirm Exon Skipping Mutations
Lourdes R. Desviat, Belén Pérez & Magdalena Ugarte
4.Analysis and Interpretation of RNA Splicing Alterations in Genes Involved in Genetic Disorders
Maaike P.G. Vreeswijk and Heleen M. van der Klift
5. Exon Skipping Mutations in Neurofibromatosis
Emanuele Buratti and Diana Baralle
Part II: Tools to Induce Exon Skipping
6. Overview on Applications of Antisense-mediated Exon Skipping
Willeke van Roon-Mom and Annemieke Aartsma-Rus
7. Overview on DMD Exon Skipping
Annemieke Aartsma-Rus
8. Overview on AON Design
Annemieke Aartsma-Rus
9. Optimizing RNA/ENA Chimeric Antisense Oligonucleotides Using in vitro Splicing
Yasuhiro Takeshima, Mariko Yagi, Masafumi Matsuo
10. Optimizing Antisense Oligonucleotides using Phosphorodiamidate Morpholino Oligomers
Linda J Popplewell, Alberto Malerba, and George Dickson
11. Optimizing Splice Switching Oligomer Sequences using 2’-O-methyl Phosphorothioate Chemistry
Carl Adkin, Sue Fletcher and Steve D Wilton
12. Exon Skipping Quantification by Real Time PCR
Alessandra Ferlini and Paola Rimessi
13. Antisense-mediated Exon Skipping to Shift Alternative Splicing to Treat Cancer
Jing Wan
14. Antisense-mediated Exon Skipping to Generate Soluble Receptors
A. Seda Yilmaz-Elis and J. Sjef Verbeek
15. Antisense-mediated Exon Skipping to Reframe Transcripts
Sandrina Turczynski, Matthias Titeux, Nathalie Pironon, and Alain Hovnanian
16. U1 snRNA as an Effective Vector for Stable Expression of Antisense Molecules andfor the Inhibition of the Splicing Reaction
Martone Julie, De Angelis Fernanda Gabriella and Bozzoni Irene
17. Engineering U7snRNA Gene to Reframe Transcripts
Aurélie Goyenvalle
18. Dynamic Fluorescent and Luminescent Reporters for Cell Based Splicing Screens
Claude C. Warzecha, Ruben Hovhannisyan, and Russ P. Carstens
19. Antisense-mediated Exon Skipping to Induce Gene Knockdown
Petra Disterer and Bernard Khoo
20. Antisense-mediated Exon Inclusion
Yimin Hua and Adrian R. Krainer
21. Antisense Genes to Induce Exon Inclusion
R. Nlend Nlend & D. Schümperli22. Using Mini-genes to Identify Factors that Modulate Alternative Splicing
Robert Morse, Adrian G. Todd and Philip J. Young
Part III: Delivery
23. Overview of Alternative Oligonucleotide Chemistries for Exon Skipping
Amer F. Saleh, Andrey A. Arzumanov and Michael J. Gait
24. Identification of Peptides for Tissue Specific Delivery
Hans Heemskerk
25. Systemic Delivery of Antisense Oligomer in Animal Models and its Implications for Treating DMD
Qi Long Lu and Bo Wu
26. Cell-penetrating Peptides Enhance Systemic Delivery of Antisense Morpholino Oligomers
Hong M Moulton
27. Optimizing Tissue-specific Antisense Oligonucleotide-peptide Conjugates
Corinne A Betts, Suzan M Hammond, Hai-fang Yin, and Matthew JA Wood
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