By Ian Gibson
Antisense and ribozymes have a comparatively brief but winning historical past as learn instruments in gene expression reviews, and therefore are regarded as having excessive capability reagents in treating viral infections and cancer.
This laboratory spouse offers specified details at the power, benefits and boundaries of this system. It seriously discusses power pitfalls, provides concepts for selecting goals and supply platforms, so that it will enable the choice of the optimal method for attaining quick and trustworthy experimental good fortune with any human or different organic system.
For researchers, technicians and complicated graduates in experimental drugs, molecular and cellphone 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
4ml for 10 min. 3. Sterilize the sample cell by treatment with 70 % alcohol. Gloves and mouth protection should be worn when transferring the solution in step 2 to the sterile cell. 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.
For short-chain sequences that correspond to short and usually chemically synthesized ribozymes, an array of systematically permutated sequences or an array of oligomeric sequences that scan along a given target sequence are fixed onto a membrane, and are subsequently hybridized with a complementary strand. , 1992). 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.
Biol. Chem. 269: 11361-6 James W, Cowe E (1996): Computational approaches to the identification of ribozyme target sites. In Ribozyme Protocols, Methods in Molecular Biology (Turner P, ed) pp 34-45, Totowa, NJ: Humana Press James H, Mills K, Gibson I (1996): Investigating and improving the specificity of ribozymes directed against the bcr-abl translocation. Leukaemia 10: 1054-64 James W, Al-Shamkhani A (1995): RNA enzymes as tools for gene ablation. Curr. Opinion Biotechnol. 6: 44-9 Kronenwett R, Haas R, Sczakiel G (1996):Kinetic selectivity of complementary nucleic acids: bcr-abl-directedantisense RNA and ribozymes.