Cardiac Cell and Gene Transfer: Principles, Protocols, and by N.J. Clifton

By N.J. Clifton

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Additional info for Cardiac Cell and Gene Transfer: Principles, Protocols, and Applications (Methods in Molecular Biology Vol 219)

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By design, gutted adenoviral vectors must be grown in the presence of a helper virus to supply in trans all the viral proteins required for growth and replication of the gutted genome. Consequently, the gutted vector must then be purified away from the helper virus that is simultaneously produced. Specific packaging cell lines may be very useful for limiting the production of infectious helper virus while promoting the growth of the gutted virus. This chapter describes the methods used by our laboratory for generating, expanding, and titering gutted adenoviral vectors for gene transfer to muscle.

3. 4. Prepare a 150-mm tissue culture dish with an 80% confluent monolayer of C7-cre cells. 5. Inoculate the plate with 2 mL of infected cell lysate (P1) supplemented with the appropriate amount of purified helper virus. The additional amount required (if any) should be based on the titer of the helper virus in P1. The final helper virus MOI should be 5–10 tu per cell. Incubate the cells until CPE is complete, usually 2–4 d. Harvest the lysate and freeze/thaw as above. Store at –70°C. 6. 3. 7. The final 2 rounds of amplification are carried out as in steps 5–6, using 10 and, then 50–100 × 150-mm dishes of C7–cre cells (see Note 7).

0, 1 mM MgCl2. This procedure is most easily accomplished using a flexible cell scraper to ensure that all the virus is retrieved. Transfer the virus solution to a 50-mL conical tube. 5. Add DNase I and RNase A to a final concentration of 50 µg/mL each. Incubate at 37°C for 30 min to remove any genomic or unpackaged nucleic acids that were coprecipitated with the virus particles. 6. 135 g CsCl per mL). When completely dissolved, pellet any residual debris by centrifuging at 8000g for 5 min at 4°C.

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