By Kiyotake Ishikawa
This designated ebook presents methodological info on cardiac gene supply, from vintage to state of the art applied sciences and strategies. effective, cardiac-specific, and secure vectors, in addition to sophisticated vector supply equipment, are key for winning cardiac gene move and at last for making improvements to sufferers’ results. more moderen vectors and extra effective vector supply tools have the capability to dramatically enhance gene transduction efficacy, whereas novel gene manipulation ideas implement the healing strength and develop affliction ambitions. Written for the hugely profitable Methods in Molecular Biology sequence, chapters contain introductions to their respective issues, lists of the required fabrics and reagents, step by step, comfortably reproducible laboratory protocols, and tips about troubleshooting and warding off recognized pitfalls.
Authoritative and useful, Cardiac Gene treatment: tools and Protocols serves as a necessary instrument for molecular biologists and physiologists within the cardiology box engaging in cardiac gene move examine, with a view to finally bring about additional developments within the very important field.
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Additional resources for Cardiac Gene Therapy: Methods and Protocols
14. 1× CIM with trypsin. Mix 10 ml 10× CIM, 80 ml H2O, and 10 ml 10× trypsin (see Note 7). 2 Materials and Equipment 1. A stack of autoclaved paper towels. 2. A small beaker glass filled with 70 % ethanol for soaking instruments. 3. A large beaker glass filled with 70 % ethanol for dipping neonate rats. 4. A spray bottle containing Barricydal or 70 % ethanol. 5. An aerosol can filled with disinfectant Barricydal or 70 % ethanol. 6. Test tube rack. 7. A bag for the unneeded parts of the animal attached to the hood for easy accessibility.
Stop cell detachment with 25 ml conditioned media from step 4 (see Note 25). 9. Transfer the suspension to 50 ml centrifuge tubes or larger centrifugation vessels if applicable. 10. Centrifuge for 20 min, 1200 × g at room temperature (see Note 26). 11. Resuspend the cell pellet in 14 ml 1× PBS and transfer it into a 50 ml centrifuge tube. 12. Centrifuge at 1200 × g for 20 min at room temperature and discard the supernatant. 13. 5 ml 1× PBS (see Note 27). 14. Disrupt the cells by 4 cycles of repeated freezing (−80 °C) and thawing (37 °C in a water bath).
22 Henry Fechner et al. 8. 500× gentamycin, 10 mg/ml: Dissolve 10 mg lyophilized sterile gentamycin in 1 ml H2O or use ready to use 10 mg/ml gentamycin solution. 9. FCS: Thaw the 500 ml bottle with frozen FCS at room temperature. Inactivate the thawed FCS (if needed) for 30 min at 56 °C using a water bath. Distribute the volume into portions of either 100 ml of 50 ml under sterile conditions and store at −20 °C. 10. Horse serum (HS): Thaw the 500 ml bottle with frozen HS at room temperature. Inactivate the thawed HS (if needed) for 30 min at 56 °C using a water bath.