Antisense and Ribozyme Methodology
Laboratory Companion
1st Edition
Published on 26. September 2008
XIII, 79 pages
978-3-527-61252-9 (ISBN)
Description
Antisense and ribozymes have a relatively short yet successful history as research tools in gene expression studies, and thus are considered as having high potential reagents in treating viral infections and cancer.
This laboratory companion provides detailed information on the potential, advantages and limitations of this methodology. It critically discusses potential pitfalls, presents strategies for choosing targets and delivery systems, so as to allow the selection of the optimum methodology for achieving fast and reliable experimental success with any human or other biological system.
For researchers, technicians and advanced graduates in experimental medicine, molecular and cell biology.
This laboratory companion provides detailed information on the potential, advantages and limitations of this methodology. It critically discusses potential pitfalls, presents strategies for choosing targets and delivery systems, so as to allow the selection of the optimum methodology for achieving fast and reliable experimental success with any human or other biological system.
For researchers, technicians and advanced graduates in experimental medicine, molecular and cell biology.
Reviews / Votes
"recommended for researchers involved in drug discovery." The Biotech Journal, April/May 2003More details
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Content
- Antisense and Ribozyme Methodology
- Contents
- CHAPTER 1. Antisense and Ribozyme Methodology
- 1.1 The Potential
- 1.2 Antisense Technology
- 1.2.1 Problems
- 1.2.2 Resistance to Nucleases
- 1.2.3 Entry into Cells
- 1.2.4 How Antisense Works
- 1.2.5 Success
- 1.3 Ribozymes
- 1.3.1 What Are They?
- 1.3.2 Problems
- 1.3.3 Stable Ribozymes
- 1.3.4 Designing Ribozymes
- 1.4 Ribozymes or Antisense DNAs?
- 1.5 The Choice Today!!
- CHAPTER 2. Design and Synthesis of Antisense DNA Molecules
- 2.1 Introduction
- 2.2 Synthesis of Methylphosphonodiester-Phosphodiester Chimeric Oligodeoxynucleotides
- 2.2.1 Materials and Chemicals
- 2.2.2 Solutions
- 2.2.3 Maximizing Product Purity
- 2.2.4 Deprotection of Chimeric Oligodeoxynucleotides
- 2.2.5 Failed Sequences
- 2.3 Primary Purification by Reversed-Phase, Solid-Phase Extraction on C18 SEP-PAK Cartridges
- 2.3.1 Equipment
- 2.3.2 Method
- 2.3.3 Purification of the Oligodeoxynucleotide
- 2.3.4 Further Purification
- 2.4 Analysis and Purification by HPLC
- 2.4.1 Analysis of Chimeric Oligodeoxynucleotides by HPLC
- 2.4.2 Purification of Chimeric Oligodeoxynucleotides by HPLC
- 2.4.3 Re-Use of Columns
- 2.5 Synthesis of Chimeric Oligodeoxynucleotides with Fluorescein Attached
- 2.6 Summary
- CHAPTER 3. The Design and Synthesis of Hammerhead Ribozymes
- 3.1 Introduction
- 3.2 The Design of Hammerhead Ribozymes
- 3.3 Improving the Reactions
- 3.3.1 Accessibility of the Target - Substrate Binding
- 3.3.2 Finding the Target
- 3.3.3 Theoretical Considerations
- 3.3.4 Experimental Approaches
- 3.3.5 Kinetic Studies
- 3.4 Length of Arms
- 3.4.1 Choosing Antisense Arms of Hammerhead Ribozymes
- 3.4.2 Arms of Different Lengths
- 3.5 Cleavage of the Target Motif
- 3.6 Synthesis of Ribozymes
- 3.6.1 Chemical Synthesis of Short Hammerhead Ribozymes
- 3.6.2 Enzymatic Transcription in Vitro
- 3.7 Endogenous Expression of Ribozyme Genes
- CHAPTER 4. Delivery of Ribozymes and Antisense DNA Molecules into Mammalian Cells
- 4.1 Introduction
- 4.2 Exogenous Application
- 4.2.1 lntracytoplasmic Delivery of Antisense Oligodeoxynucleotides by Reversible Plasma Membrane Permeabilization with Streptolysin O
- 4.3 Microinjection
- 4.4 Other Methods Used in Nucleic Acid Transfection
- 4.5 Electroporation
- 4.5.1 Method
- 4.5.2 Transfection: Optimization of Conditions
- 4.5.3 Mechanism of Uptake Following Electroporation
- 4.5.4 Benefits and Drawbacks of Electrophoretic-Mediated Uptake
- 4.6 Diethylaminoethyl-Dextran (DEAE- Dextran) and DNA Transfection
- 4.6.1 Methods for Transfection of Adherent Cells
- 4.6.2 Possible Alterations of the Above Protocol
- 4.6.3 Transfection of Cells Growing in Suspension
- 4.6.4 Transfection Optimization
- 4.6.5 Distribution Mechanism of DEAE-Dextran Uptake and lntracellular
- 4.6.6 Benefits and Drawbacks
- 4.7 Calcium Phosphate Transfection
- 4.7.1 Method
- 4.7.2 Possible Alterations to Above Method
- 4.7.3 Method Optimization
- 4.7.4 Calcium Phosphate-Mediated Uptake and lntracellular Distribution
- 4.7.5 Benefits and Drawbacks of Calcium Phosphate-Mediated Uptake
- 4.8 Cationic Lipids
- 4.8.1 Cationic Lipid Formulations
- 4.8.2 Methods
- 4.8.3 Lipofectin-Mediated Transient Transfection of Adherent Cells
- 4.8.4 Lipofectin-Mediated Stable Transfection of Adherent Cells
- 4.8.5 Lipofectin-Mediated Transfection of Cells insuspension
- 4.8.6 Lipofectamine-Mediated Transient or Stable Transfection of Adherent Cells
- 4.8.7 Lipofectamine-Mediated Transfection of Cells insuspension
- 4.8.8 Optimizing Transfection
- 4.8.9 Cationic Lipid Uptake and lntracellular Distribution
- 4.8.10 Benefits and Drawbacks of Cationic Lipids
- 4.8.11 Future Developments
- 4.9 Vector-Mediated Delivery
- 4.10 Conclusions
- CHAPTER 5. The Future
- Appendix
- Subject Index
