By Ian Gibson
Antisense and ribozymes have a comparatively brief but winning heritage as learn instruments in gene expression stories, and hence are regarded as having excessive capability reagents in treating viral infections and cancer.
This laboratory better half offers certain info at the capability, benefits and boundaries of this technique. It seriously discusses strength pitfalls, offers recommendations for selecting goals and supply platforms, with a purpose to permit the choice of the optimal technique for reaching speedy and trustworthy experimental good fortune with any human or different organic system.
For researchers, technicians and complex graduates in experimental drugs, molecular and phone biology.
Chapter 1 Antisense and Ribozyme method (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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Additional resources for Antisense and Ribozyme Methodology: Laboratory Companion
Br. J. Cancer 60: 343-50 Toulme J-J, Tidd D M (1997): The role of ribonuclease H in antisense oligonucleotide-mediated effects. In Ribonuclease H (Crouch RJ, Toulme J-J, eds) Paris: Les Editions INSERM, In press Zon G (1993):Oligonucleoside phosphorothioates. In Methods in Molecular Biology, Vol. 20, Protocols for oligonucleotides and analogs. Synthesis and properties (Agrawal S, ed) pp 165-89, Totowa, NJ: Humana Press Design and Synthesis of Antisense DNA Molecules Antisense and Ribozyme Methodology Ian Gibson Cowriaht 02002 WILEY-VCH Verlaa GmbH & Co.
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. , 1987). The main parameters which should be optimized for each cell line are field strength, number of pulses, length of intervals, incubation period before addition of complete medium, and DNA concentration. Winterbourne et a/. (1988)found field strength and pulse duration to be the critical parameters.
The observed variations of the cleavage efficiencies may be partially explained by differences of the neighboring sequences, which were not the same among the constructs compared in Fig. 4. , 1990 Fig. 7 Cleavage efficiency of base triplets by hammerhead ribozyme on the cleavage efficiency, which is stronger than the influence of bases downstream (3’). O n the other hand, a recent study by Shimayama ei a/. (1995) showed that even in the presence of mismatches in the NUX motif, cleavage was still surprisingly efficient.