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Antisense and Ribozyme Methodology

Overview

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...
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Overview

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.

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Editorial Reviews

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"...recommended for researchers involved in drug discovery." (The Biotech Journal, April/May 2003)
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Product Details

  • ISBN-13: 9783826100796
  • Publisher: Chapman & Hall
  • Publication date: 9/1/1997
  • Series: Laboratory Companion Ser.
  • Format: Mass Market Paperback
  • Pages: 70

Table of Contents

Chapter 1. Antisense and Ribozyme Methodology 1
1.1 The Potential 1
1.2 Antisense Technology 4
1.2.1 Problems 4
1.2.2 Resistance to Nucleases 4
1.2.3 Entry into Cells 5
1.2.4 How Antisense Works 6
1.2.5 Success 6
1.3 Ribozymes 7
1.3.1 What Are They? 7
1.3.2 Problems 8
1.3.3 Stable Ribozymes 8
1.3.4 Designing Ribozymes 8
1.4 Ribozymes or Antisense DNAs? 9
1.5 The Choice Today!! 10
Chapter 2. Design and Synthesis of Antisense DNA Molecules 13
2.1 Introduction 13
2.2 Synthesis of Methylphosphonodiester-Phosphodiester Chimeric Oligodeoxynucleotides 15
2.2.1 Materials and Chemicals 15
2.2.2 Solutions 15
2.2.3 Maximizing Product Purity 16
2.2.4 Deprotection of Chimeric Oligodeoxynucleotides 16
2.2.5 Failed Sequences 17
2.3 Primary Purification by Reversed-Phase, Solid-Phase Extraction on C18 SEP-PAK Cartridges 17
2.3.1 Equipment 17
2.3.2 Method 18
2.3.3 Purification of the Oligodeoxynucleotide 18
2.3.4 Further Purification 19
2.4 Analysis and Purification by HPLC 20
2.4.1 Analysis of Chimeric Oligodeoxynucleotides by HPLC 20
2.4.2 Purification of Chimeric Oligodeoxynucleotides by HPLC 21
2.4.3 Re-Use of Columns 22
2.5 Synthesis of Chimeric Oligodeoxynucleotides with Fluorescein Attached 23
2.6 Summary 25
Chapter 3. The Design and Synthesis of Hammerhead Ribozymes 27
3.1 Introduction 27
3.2 The Design of Hammerhead Ribozymes 29
3.3 Improving the Reactions 29
3.3.1 Accessibility of the Target--Substrate Binding 29
3.3.2 Finding the Target 30
3.3.3 Theoretical Considerations 30
3.3.4 Experimental Approaches 31
3.3.5 Kinetic Studies 31
3.4 Length of Arms 33
3.4.1 Choosing Antisense Arms of Hammerhead Ribozymes 33
3.4.2 Arms of Different Lengths 33
3.5 Cleavage of the Target Motif 34
3.6 Synthesis of Ribozymes 35
3.6.1 Chemical Synthesis of Short Hammerhead Ribozymes 36
3.6.2 Enzymatic Transcription in Vitro 36
3.7 Endogenous Expression of Ribozyme Genes 37
Chapter 4. Delivery of Ribozymes and Antisense DNA Molecules into Mammalian Cells 41
4.1 Introduction 41
4.2 Exogenous Application 41
4.2.1 Intracytoplasmic Delivery of Antisense Oligodeoxy-nucleotides by Reversible Plasma Membrane Permeabilization with Streptolysin O 43
4.3 Microinjection 44
4.4 Other Methods Used in Nucleic Acid Transfection 45
4.5 Electroporation 45
4.5.1 Method 45
4.5.2 Transfection: Optimization of Conditions 46
4.5.3 Mechanism of Uptake Following Electroporation 46
4.5.4 Benefits and Drawbacks of Electrophoretic-Mediated Uptake 47
4.6 Diethylaminoethyl-Dextran (DEAE-Dextran) and DNA Transfection 47
4.6.1 Methods for Transfection of Adherent Cells 47
4.6.2 Possible Alterations of the Above Protocol 48
4.6.3 Transfection of Cells Growing in Suspension 49
4.6.4 Transfection Optimization 50
4.6.5 Mechanism of DEAE-Dextran Uptake and Intracellular Distribution 51
4.6.6 Benefits and Drawbacks 51
4.7 Calcium Phosphate Transfection 51
4.7.1 Method 52
4.7.2 Possible Alterations to Above Method 53
4.7.3 Method Optimization 54
4.7.4 Calcium Phosphate-Mediated Uptake and Intracellular Distribution 55
4.7.5 Benefits and Drawbacks of Calcium Phosphate-Mediated Uptake 56
4.8 Cationic Lipids 57
4.8.1 Cationic Lipid Formulations 57
4.8.2 Methods 58
4.8.3 Lipofectin-Mediated Transient Transfection of Adherent Cells 59
4.8.4 Lipofectin-Mediated Stable Transfection of Adherent Cells 59
4.8.5 Lipofectin-Mediated Transfection of Cells in Suspension 60
4.8.6 Lipofectamine-Mediated Transient or Stable Transfection of Adherent Cells 60
4.8.7 Lipofectamine-Mediated Transfection of Cells in Suspension 62
4.8.8 Optimizing Transfection 62
4.8.9 Cationic Lipid Uptake and Intracellular Distribution 64
4.8.10 Benefits and Drawbacks of Cationic Lipids 65
4.8.11 Future Developments 66
4.9 Vector-Mediated Delivery 67
4.10 Conclusions 67
Chapter 5. The Future 73
Appendix 77
Subject Index 79
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