Editing Adenovirus AdenoX (section)

== <br>Amplifying Recombinant Adenovirus: Preparing High-Titer Stocks ==

<br>Note: The probability of producing replication competent adenovirus (RCA), although low, increases with each successive amplification. RCA is produced when Adeno-X DNA recombines with E1-containing genomic DNA in 293 cells. For this reason, we suggest you save aliquots of early amplifications. Use early amplification stocks whenever you need to produce additional quantities of adenovirus.

1. About 24 hours before infection, plate HEK 293 cells in a T75 flask. The cell monolayer should be 50â70% confluent when you infect.

2. Incubate cells overnight at 37°C in a humidified atmosphere maintained at 5% CO2.

3. On the following day, replace the medium with 5 ml of fresh growth medium that contains adenovirus: For best results, infect cells at a multiplicity of â¥5 (i.e., at â¥5 pfu/cell). For example, if the T75 flask contains ~5 x 106 cells, add 2.5 x 107 pfu adenovirus.

4. Incubate for 90 min at 37°C in a humidified atmosphere maintained at 5% CO2.

5. Remove the flask and add 10 ml of fresh growth medium.

6. Incubate for 3â4 days at 37°C in a humidified atmosphere at 5% CO2.

7. Check for a cytopathic effect. When 50% of the cells have detached, transfer the suspension to a sterile 15-ml conical centrifuge tube.<br>Do not use trypsin: Infected cells that still adhere to the bottom or sides of the flask can be dislodged into the medium by gentle agitation.

8. Isolate virus using the freeze-thaw method given in Part B, Steps 5â9. (At Step B.7, resuspend the pellet in 0.5â1 ml of PBS.)

9. Determine adenoviral titer (Appendix D). We recommend the Adeno-Xâ¢ Rapid Titer Kit (Cat. No. 631028). Expected titer: 10^8â10^9 pfu/ml.

10. To produce a greater quantity of high-titer adenovirus, use the cell lysate from the first amplification to infect larger cultures (e.g., a series of T175 flasks).

11. [Optional] Depending on how you intend to use your recombinant adenovirus, you may wish to refine your High-Titer Stock by banding on a CsCl density gradient. Please consult the following references for protocols on how to purify adenovirus using CsCl gradient centrifugation (Hitt et al., 1998; Hitt et al., 1995; Graham &amp; Prevec, 1991; Spector &amp; Samaniego, 1995; and Becker et al., 1994).

Alternatively, use the Adeno-X Virus Purification Kit (Cat. Nos. 631532, 631533 &amp; 631534). With this kit, adenovirus can be purified in less than 2 hours, and no ultracentrifugation steps are necessary.

<br>The purity is comparable to that achieved with CsCl centrifugation. See the July 2002 issue of Clontechniques, available from Clontech web site, or download a free copy of the User Manual (PT3680-1) to learn more about this product.<br>

[[Category:Protocols]]