By Ian Gibson
Antisense and ribozymes have a comparatively brief but profitable background as study instruments in gene expression experiences, and hence are regarded as having excessive power reagents in treating viral infections and melanoma. This laboratory significant other presents targeted info at the power, merits and obstacles of this technique. It seriously discusses capability pitfalls, provides options for selecting goals and supply platforms, with the intention to let the choice of the optimal method for reaching speedy and trustworthy experimental good fortune with any human or different organic approach. For researchers, technicians and complicated graduates in experimental drugs, molecular and cellphone biology.
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Extra resources for Antisense and Ribozyme Methodology: Laboratory Companion (Laboratory Companion Series)
Nucl. Acids Res. 21 : 5171-8 Forster AC, Symons RH (1987):Self cleavage of plus and minus RNAs of a virusoid and a structural model for the active site. Cell 49: 211-20 Haseloff J, Gerlach WL (1988):Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature 334: 585-91 Heinrich J-C, Tabler M, Louis C (1993):Attenuation of white gene expression in transgenic Drosophila melanogaster: Possible role of a catalytic antisennse RNA. Dev. Genet. 14: 258-65 Hernandez C, Flores R (1992):Plus and minus RNAs of peach latent mosaic viroid self-cleavage in vitro via hammerhead structures.
The partially purified chimeric oligodeoxynucleotide product is eluted from the cartridge with 3 ml 50 % acetonitrile/water (1:1, v/v), and the total number of A260units recovered is determined from the absorbance at 260 nm of a diluted sample. Samples of the eluate are analyzed by weak anion exchange HPLC and by reverse phase HPLC (see below). A certain degree of cleavage of methylphosphonodiester internucleosi linkages may occur during deprotection, to yield fragments carrying a 5’-dimethoxytrityl group which will copurify with the product during trityl selection on C18 SepPak cartridges.
Addition to the culture medium or tissue, allows continuous delivery of ribozymes or antisense DNA molecules, but is restricted to small modified ribozymes or antisense molecules that are sufficiently stable and membrane permeable. For long-chain ribozymes and ribozyme encoding genes, however, this approach is regarded as inappropriate. One convincing study, however, involves the delivery of small antisense DNA molecules to cells following streptolysin treatment of the cells. , addition to culture medium uptake of oligonucleotides I.