LEADER 05251nam 2200661 450 001 9910830630503321 005 20230421044236.0 010 $a1-281-84239-7 010 $a9786611842390 010 $a3-527-61253-X 010 $a3-527-61252-1 035 $a(CKB)1000000000555626 035 $a(EBL)482019 035 $a(OCoLC)291090796 035 $a(SSID)ssj0000104258 035 $a(PQKBManifestationID)11131019 035 $a(PQKBTitleCode)TC0000104258 035 $a(PQKBWorkID)10078050 035 $a(PQKB)10445682 035 $a(MiAaPQ)EBC482019 035 $a(EXLCZ)991000000000555626 100 $a20160819h19971997 uy 0 101 0 $aeng 135 $aur|n|---||||| 181 $ctxt 182 $cc 183 $acr 200 00$aAntisense and ribozyme methodology /$fIan Gibson, editor 210 1$aLondon, [England] :$cChapman & Hall,$d1997. 210 4$dİ1997 215 $a1 online resource (96 p.) 225 1 $aLaboratory Companion Series 300 $aIncludes index. 300 $a"With 10 Figures, some in Color"--Title page. 311 $a3-8261-0079-4 311 $a3-527-30889-X 327 $aAntisense 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 327 $a2.2.1 Materials and Chemicals2.2.2 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 327 $a2.5 Synthesis of Chimeric Oligodeoxynucleotides with Fluorescein Attached2.6 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 327 $a3.6.1 Chemical Synthesis of Short Hammerhead Ribozymes3.6.2 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 327 $a4.5.3 Mechanism of Uptake Following Electroporation4.5.4 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 327 $a4.7.4 Calcium Phosphate-Mediated Uptake and lntracellular Distribution 330 $aAntisense and ribozymes have a relatively short yet successful history as research tools in gene expression studies, and thus are considered as having high potential reagents in treating viral infections and cancer.This laboratory companion provides detailed information on the potential, advantages and limitations of this methodology. It critically discusses potential pitfalls, presents strategies for choosing targets and delivery systems, so as to allow the selection of the optimum methodology for achieving fast and reliable experimental success with any human or other biological system. 410 0$aLaboratory companion. 606 $aAntisense nucleic acids$vLaboratory manuals 606 $aCatalytic RNA$vLaboratory manuals 615 0$aAntisense nucleic acids 615 0$aCatalytic RNA 676 $a572.88 676 $a615.36 702 $aGibson$b Ian$f1938- 801 0$bMiAaPQ 801 1$bMiAaPQ 801 2$bMiAaPQ 906 $aBOOK 912 $a9910830630503321 996 $aAntisense and ribozyme methodology$91933177 997 $aUNINA