Cardiac Gene Therapy: Methods and Protocols by Kiyotake Ishikawa
By Kiyotake Ishikawa
This precise ebook presents methodological info on cardiac gene supply, from vintage to state of the art applied sciences and methods. effective, cardiac-specific, and secure vectors, in addition to sophisticated vector supply equipment, are key for profitable cardiac gene move and at last for bettering sufferers’ results. more recent vectors and extra effective vector supply tools have the capability to dramatically enhance gene transduction efficacy, whereas novel gene manipulation ideas implement the healing energy and expand sickness objectives. 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 averting identified pitfalls.
Authoritative and functional, Cardiac Gene remedy: equipment and Protocols serves as a worthy device for molecular biologists and physiologists within the cardiology box carrying out cardiac gene move examine, with the intention to eventually result in additional developments within the very important field.
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Extra info for Cardiac Gene Therapy: Methods and Protocols
Centrifuge 15 min at 10,000 × g and transfer the supernatant into a 15 ml centrifuge tube. 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.
4 Notes 1. Using this procedure the volume of the solution will be increased by 150 ml. 2. Do not store this glucose containing solution longer than 3 days at 4 °C. 3. Most commercially available trypsin preparations for cell cultures purposes will not work properly for neonatal cardiomyocyte preparation. Best results can be obtained with crude trypsin preparations, although not all crude enzyme prepara- 34 Henry Fechner et al. tions will work properly and vary from lot to lot. Therefore, it is necessary to prescreen several lots of crude trypsin before selecting a lot to purchase.
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. 6. Resuspend the pellet in 5 ml NaCl–Hepes solution. 7. Centrifuge 15 min at 10,000 × g and transfer the supernatant into a 15 ml centrifuge tube. 8. Add 50 μl Benzonase Nuclease to the supernatant (final concentration 250 U/ml) and incubate for 1 h at 37 °C.