By Ian Gibson
Antisense and ribozymes have a comparatively brief but winning background as learn instruments in gene expression experiences, and hence are regarded as having excessive strength reagents in treating viral infections and cancer.
This laboratory better half offers certain info at the capability, merits and obstacles of this technique. It significantly discusses strength pitfalls, provides techniques for selecting ambitions and supply structures, in an effort to enable the choice of the optimal technique for attaining quickly and trustworthy experimental luck with any human or different organic system.
For researchers, technicians and complex graduates in experimental medication, molecular and phone biology.
Chapter 1 Antisense and Ribozyme technique (pages 1–12):
Chapter 2 layout and Synthesis of Antisense DNA Molecules (pages 13–26):
Chapter three The layout and Synthesis of Hammerhead Ribozymes (pages 27–40):
Chapter four supply of Ribozymes and Antisense DNA Molecules into Mammalian Cells (pages 41–71):
Chapter five the long run (pages 73–76):
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Extra info for Antisense and Ribozyme Methodology: Laboratory Companion
Nature 328: 596-600 Zoumadakis M, Tabler M (1995):Comparative analysis of cleavage rates after systematic permutation of the NUX0 consensus target motif for hammerhead ribozymes. Nucl. Acids Res. 23: 1192-6 Zoumadakis M, Neubert WJ, Tabler M (1994):The influence of imperfectly paired helices I and Ill on the catalytic activity of hammerhead ribozymes. Nucl. Acids Res. 22: 5271-8 References Antisense and Ribozyme Methodology Ian Gibson Cowriaht 02002 WILEY-VCH Verlaa GmbH & Co. 1 Introduction A variety of methods have been used to deliver ribozyme and antisense DNA molecules into living cells (Fig.
4. Three successive pulses are applied at an interval of 3 sec. The sample is kept at 20 "C at a field strength of 8 k 0 . 5 kV/cm. 5. Ten minutes after the electrical impulses, aliquots of 100 pl of cell suspension are transferred to DMEM. 6. After 24 h, incubate the cell monolayers in HAT (Hypoxanthine, Aminopterin and Thymidine) medium under standard conditions. 2 Transfection: Optimization of Conditions parameters to be optimized Although Neumann et al. (1982)recommend optimizing the technique for each cell line, and especially for cell size, it is noted that even for non-optimized conditions the yield obtained is comparable to other methods.
5 Kinetic Studies For long-chain sequences (< nt), an alternative procedure has been developed. 3 Improving the Reactions Fig. Briefly, a parental antisense RNA or ribozyme, preferentially an asymmetric hammerhead ribozyme, is end-labeled and subjected to limited alkaline hydrolysis, thereby generating a pool of successively shortened and end-labeled antisense or ribozyme species. This pool is incubated together with a large excess of target RNA in sohtion, and aliquots are withdrawn to follow the time course of the annealing reaction.
Antisense and Ribozyme Methodology: Laboratory Companion by Ian Gibson