By Kiyotake Ishikawa
This exact publication presents methodological info on cardiac gene supply, from vintage to cutting-edge applied sciences and methods. effective, cardiac-specific, and secure vectors, in addition to subtle vector supply tools, are key for profitable cardiac gene move and finally for making improvements to sufferers’ results. more moderen vectors and extra effective vector supply tools have the aptitude to dramatically enhance gene transduction efficacy, whereas novel gene manipulation thoughts implement the healing energy and increase disorder goals. Written for the hugely profitable Methods in Molecular Biology sequence, chapters contain introductions to their respective themes, lists of the required fabrics and reagents, step by step, comfortably reproducible laboratory protocols, and tips about troubleshooting and fending off recognized pitfalls.
Authoritative and sensible, Cardiac Gene remedy: equipment and Protocols serves as a worthy software for molecular biologists and physiologists within the cardiology box carrying out cardiac gene move learn, so as to finally result in extra developments within the very important field.
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Extra info for Cardiac Gene Therapy: Methods and Protocols
4 Precipitation of AAV Vectors from Supernatant (see Note 29) 1. 5 M CaCl2 per 100 ml supernatant, mix and cool on ice for 1 h. 2. Distribute the solution into 50 ml centrifuge tubes or larger centrifugation vessels. AAV Vectors for Cardiac Gene Silencing 29 3. Centrifuge for 30 min at 3900 × g and transfer the supernatant into a 400 ml Erlenmeyer flask. 4. 86 M NaCl/24 % PEG per 100 ml supernatant, mix and incubate for 72 h at 4 °C. 5. Centrifuge for 30 min at 3000 × g and discard the supernatant.
34. If solution remains in the tube and does not pass through (which is a result of remaining iodixanol in the preparation), resuspend and rinse the membrane of the Amicon® Ultra 15 ml centrifugal filters with the remaining volume or extend centrifugation time. 35. For large volumes of AAV, several Amicon® Ultra 15 ml centrifugal filters can be used in parallel. After centrifugation the concentrated AAV solutions can be combined and concentrated further using a fresh Amicon® Ultra 15 ml centrifugal filter.
Centrifuge for 20 min at 3900 × g and transfer the supernatant to a new 15 ml centrifuge tube. Mix the supernatant 1:1 with 1× PBS. The final volume is about 10 ml (see Note 30). 5 Filtration of AAV Vectors with Iodixanol Gradient System 1. Prepare iodixanol dilutions as shown in Table 2. 2. 5 ml 15 % iodixanol to a 22 ml polypropylene centrifuge tube. 3. 5 ml 40 % iodixanol and finally with 6 ml 54 % iodixanol using a long cannula. 4. 4 (see Note 31). 5. Fill-up the tube with PBS-MK to the mark on the tube (see Note 32).