By Jeffrey R. Morgan
During this absolutely up-to-date and revised 2d version of Gene remedy Protocols, best specialists from educational and commercial laboratories all over the world aspect their top-rated viral and nonviral equipment of gene move, in addition to speak about their purposes in several organ structures. those tools variety from these during which new molecular conjugates express nice promise for focusing on gene move and regulating transgene expression, to these utilized in such intriguing purposes because the supply of healing proteins, vaccination, and tissue engineering. up to date and hugely sensible, Gene remedy Protocols, 2d variation, bargains a wealthy compilation of the progressive advances that experience lately happened in gene move expertise, with every one article delivering confirmed the step by step laboratory techniques that permit its winning healing program.
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Extra resources for Gene Therapy Protocols 2nd Edition (Methods in Molecular Medicine)
2. 3. 4. Polylysine (PL)(MW 3970) HBr. Polyethylene glycol (PEG)-succinyl ester (MW 5000). Potassium sulfate. Dimethyl sulfoxide (DMSO). Targeted Gene Transfer to Liver Using Protein DNA Complexes 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 17 Dithiothreitol (DTT). Sodium chloride (NaCl). Sodium acetate. Lysine ester. Sodium hydroxide (NaOH). Sodium dodecyl sulfate (SDS). Ammonium bicarbonate (NH4HCO3). Ethylenediamine tetraacetic acid (EDTA). Ethidium bromide.
Inoculate a stab from a frozen culture of L. monocytogenes into 15 mL of Brain Heart Infusion medium and incubate with shaking overnight at 37°C. 2. Add the overnight culture to 1 L of Luria-Bertani (LB) broth, which is prewarmed to 37°C. 3. We recommend growing 6 L per purification batch, each grown for 15 h. 4. Remove bacteria by centrifugation at 10,000g for 15 min at 4°C. 5. Filter the supernatant through Whatman #1 filter paper, keeping the receiving flask on ice. 6. Apply 6 L of chilled supernatant to a CH2 spiral cartridge concentrater with a S1Y30 spiral cartridge of 30,000 mol wt cut-off, concentrate to 500 mL.
2:1. 6. Modify the conjugate with SPDP to introduce DTP groups for conjugating with VSV peptide. 7. 7. Monitor the conjugation of Targeted Gene Transfer to Liver Using Protein DNA Complexes 21 PEG-PL-DTP with VSV by measuring the absorption at 343 nm, because of release of 2-mercaptopyridine (9). 8. 6 (10; see Note 4). 4. Measurement of DNA Binding and Compaction To assess compaction of DNA after complexation with various conjugates, fluorescence of ethidium bromide excluded from DNA complexes was used.