By Ian Gibson
Antisense and ribozymes have a comparatively brief but profitable heritage as examine instruments in gene expression experiences, and hence are regarded as having excessive power reagents in treating viral infections and cancer.
This laboratory significant other presents specified details at the capability, benefits and barriers of this system. It significantly discusses power pitfalls, provides techniques for selecting objectives and supply platforms, as a way to permit the choice of the optimal technique for attaining quick and trustworthy experimental luck with any human or different organic system.
For researchers, technicians and complex graduates in experimental medication, molecular and mobilephone 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 longer term (pages 73–76):
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Additional resources for Antisense and Ribozyme Methodology: Laboratory Companion
EM60 J. 11: 1913-19 Perriman R, Delves A, Gerlach WL (1992): Extended target-site specificity for a hammerhead ribozyme. Gene 113: 157-63 Prody GA, Bakos JT, Buzayan JM etal. (1986):Autolytic processing of dimeric plant virus satellite RNA. Science 231: 1577-80 Rhodes A, James WJ (1990) Inhibition of human immunodeficiency virus replication by endogenously synthesized antisense RNA. J. Gen. Virol. 71: 1965-74 The Design and Synthesis of Hammerhead Ribozymes Rittner K, Burmester C, Sczakiel G (1993):In vitro selection of fast-hybridizing and effective antisense RNAs directed against the human immunodeficiency virus type 1.
Opinion Biotechnol. 6: 44-9 Kronenwett R, Haas R, Sczakiel G (1996):Kinetic selectivity of complementary nucleic acids: bcr-abl-directedantisense RNA and ribozymes. J. Mol. Biol. 259: 632-44 Kuo MYP, Sharmeen L, Dinter-Gottlieb D et al. (1988): Characterization of selfcleaving RNA sequences on the genome and antigenome of human hepatitis delta virus. J. Virol. 62: 2674-9 Lieber A, Strauss M (1995):Selection of efficient cleavage sites in target RNA by using a ribozyme expression library. Mol. Cell.
The capping step is omitted by jumping to the next command, and 2further cycles are executed for coupling of an arbitrary deoxynucleoside phosphoramidite. The trityl fraction of the first supplementary cycle, which ideally should be colorless, gives an indication of the efficiency of coupling of amino-linker. If coupling has been less than ideal, the detritylation step of the final cycle may be omitted, as for trityl-on oligodeoxynucleotide synthesis. 5 Synthesis of Chimeric Oligodeoxynucleotides with Fluorescein Attached problems in separation coupling of fluorescein removed during initial C18 Sep-Pak clean-up of deprotected product, through a reversal of the strategy normally used to select trityl-on product from failure sequences.
Antisense and Ribozyme Methodology: Laboratory Companion by Ian Gibson