Adenovirus AdenoX

Transfecting HEK 293 Cells with Pac I-Digested Adeno-X™ DNA

1. Plate HEK 293 cells at a density of 1–2 x 106 cells per 60-mm culture plate (approximately 100 cells/mm2) 12–24 hr before transfection.
For best results, cells should be 50–70% confluent, display a flat morphology, and adhere well to the plate just prior to transfection.

If you constructed a positive control vector (e.g., pAdeno-X-lacZ), be sure to seed sufficient plates to produce this virus as well. Transfection control: To monitor the efficiency of your transfection procedure, transfect an additional plate with pShuttle2-lacZ. Approximately 48 hr after transfection, check for the expression of β-galactosidase by staining cells with X-Gal.

2. Incubate the plate(s) at 37°C in a humidified atmosphere maintained at 5% CO2.
3. Transfect each 60-mm culture plate with 10 ÎŒl of Pac I-digested Adeno-X DNA. Use any GeneExpressoMax transfection method to transfer DNA into HEK 293 cells.

4. One day later, and periodically thereafter, check for cytopathic effect (CPE).

Notes:
• Infected cells typically remain intact but round up and may detach from the plate.

These changes are collectively referred to as the cytopathic effect (CPE). For a description of the CPE, please see the Adeno-X™ Frequently Asked Questions at www.clontech.com/clontech/techinfo/faqs/.

• The time it takes for CPE to appear depends on the transfection efficiency—it may take up to two weeks for CPE to become evident. Because adenovirus remains associated with cells until late in the infection cycle, high titer virus is obtained by manually lysing cells with a series of freeze-thaw cycles as explained below in Steps 5–9.

5. One week later, transfer cells to a sterile 15-ml conical centrifuge tube. Do not use trypsin: Infected cells that still adhere to the bottom or sides of the culture plate can be dislodged into the medium by gentle agitation.

6. Centrifuge the suspension at 1,500 x g for 5 min at room temperature.

7. Resuspend the pellet in 500 ÎŒl sterile PBS.

8. Lyse cells with three consecutive freeze-thaw cycles: Freeze cells in a dry ice/ethanol bath; thaw cells by placing the tube in a 37°C water bath. Do not allow the suspension to reach 37°C. Vortex cells
after each thaw.

9. After the third cycle, briefly centrifuge to pellet debris. Transfer the lysate to a clean, sterile centrifuge tube and store the lysate at –20°C or use immediately for Step 10.

10. Infect a fresh 60-mm culture by adding 250 ÎŒl (50%) of the cell lysate from Step 9. Add the lysate directly to the medium, then incubate as normal. CPE should be evident within one week.

Note: If no CPE appears after one week, the viral titer of the cell lysate from Step 9 may be too low. Amplify the titer by repeating Steps 5–10.

11. When >50% of the cells have detached from the plate, prepare viral stock by following Steps 5–9. Name this stock “Primary Amplification”. Store at –20°C.

• Primary Amplification Stock is suitable for infecting target cells. The function of this viral stock should be evaluated before preparing High-Titer Stock (Part C).
• The presence of your recombinant construct can be verified by PCR or Western blotting (Part D) .

12. Determine adenoviral titer (Appendix D).

The Adeno-X™ Rapid Titer Kit (Cat. No. 631028) enables you to determine adenoviral titer using an anti-hexon antibody cell staining assay. See the April 2002 issue of Clontechniques, available from Clontech web site, or download a free copy of the User Manual (PT3651-1) to learn more about this product.

Amplifying Recombinant Adenovirus: Preparing High-Titer Stocks

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 50–70% 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 ≥5 (i.e., at ≥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 3–4 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.
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 5–9. (At Step B.7, resuspend the pellet in 0.5–1 ml of PBS.)

9. Determine adenoviral titer (Appendix D). We recommend the Adeno-X™ Rapid Titer Kit (Cat. No. 631028). Expected titer: 10^8–10^9 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 & Prevec, 1991; Spector & Samaniego, 1995; and Becker et al., 1994).

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

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.