By Kiyotake Ishikawa
This exact publication offers methodological details on cardiac gene supply, from vintage to cutting-edge applied sciences and methods. effective, cardiac-specific, and secure vectors, in addition to sophisticated vector supply equipment, are key for winning cardiac gene move and finally for bettering sufferers’ results. more moderen vectors and extra effective vector supply equipment have the aptitude to dramatically enhance gene transduction efficacy, whereas novel gene manipulation recommendations implement the healing strength and increase sickness pursuits. Written for the hugely profitable Methods in Molecular Biology sequence, chapters comprise introductions to their respective subject matters, lists of the mandatory fabrics and reagents, step by step, without problems reproducible laboratory protocols, and tips about troubleshooting and warding off recognized pitfalls.
Authoritative and functional, Cardiac Gene remedy: tools and Protocols serves as a important software for molecular biologists and physiologists within the cardiology box undertaking cardiac gene move study, in an effort to finally result in extra developments within the very important field.
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Extra resources for Cardiac Gene Therapy: Methods and Protocols
You can also use standard disposable cell culture vessels (flasks or plates) for production. 5 cm diameter). 19. The collagen is available from Sigma-Aldrich (Cat. No. C8919). html#sthash. dpuf 20. Removed collagen solution can be reused. We have reused it for up to 10 times. Coated roller bottles can be stored at 4 °C for several days. 21. In each step when a solution is added/removed from the roller bottle set down the bottle gently to avoid shaking off cells. Also avoid spraying added solutions onto the walls of the bottle where cells are growing, as this may lead to unintentional detachment of the cells.
8. Add 50 μl Benzonase Nuclease to the supernatant (final concentration 250 U/ml) and incubate for 1 h at 37 °C. Mix from time to time. 9. 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.
Incubate the cell culture plates for 24 h at 37 °C in an incubator (see Note 45). 17. 7, step 15) of complete CMRL1415-ATM cell culture medium with 10 % FCS containing also 2 μM FUDR in addition to 20 μg/ml gentamycin (see Note 46). 18. After another 48 h of incubation replace the medium by fresh CMRL1415-ATM cell culture medium with 10 % FCS, 2 μM FUDR, and 20 μg/ml gentamycin and transduce the cells with scAAV vectors by directly adding the vector into the cell culture medium and incubate at 37 °C as before (see Note 47).