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 strength reagents in treating viral infections and cancer.
This laboratory better half presents specific details at the power, benefits and barriers of this system. It seriously discusses capability pitfalls, provides ideas for selecting ambitions and supply platforms, on the way to let the choice of the optimal technique for reaching quickly and trustworthy experimental luck 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 long run (pages 73–76):
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Additional info for Antisense and Ribozyme Methodology: Laboratory Companion
A cDNA fragment serving for substrate binding is selected, and two primers for PCR are synthesized, such that the 5'-primer contains the sequences that form three base pairs in helix I of the hammerhead, as well as the catalytic The Design and Synthesis of Hammerhead Ribozymes domain (Fig. 6). The 3’-primer is a simple short primer that defines the length of helix Ill. The resulting PCR products can be used to synthesize RNA directly or can be used for the cloning of recombinant ribozyme genes (see below).
1 Method The following method is as optimized by Neumann et al. (1982)for the transfection of both circular and linear DNA into mouse LTK cells. 1. 1; at 20 "C) to a final concentration of 5 x lo7 cells/ml. 5 Electroporation 2. 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 .
The following is an example of an experiment to determine the optimum streptolysin 0 concentration. use of streptolysin preparation of streptolysin 0 solution optimization of permeabilization 1. 1 ml of permeabilization buffer containing streptolysin 0 at a range of concentrations, and fluorescein-tagged oligodeoxynucleotide at 1 p ~The . optimum concentration of streptolysin 0 may vary over the range of 10-200 U/ml, depending on the batch. 2. The suspensions are incubated at 37 "C for 20 min before resealing is induced by addition of 1 ml tissue culture medium containing 10 % fetal bovine serum.