Editing Adenovirus AdenoX (section)

== Transfecting HEK 293 Cells with Pac I-Digested Adeno-Xâ¢ DNA ==

1. Plate HEK 293 cells at a density of 1â2 x 106 cells per 60-mm culture plate (approximately 100 cells/mm2) 12â24 hr before transfection.<br>For best results, cells should be 50â70% 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 Î²-galactosidase by staining cells with X-Gal.

2. Incubate the plate(s) at 37°C in a humidified atmosphere maintained at 5% CO2.<br>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:'''<br>â¢ 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-Xâ¢ Frequently Asked Questions at www.clontech.com/clontech/techinfo/faqs/.

â¢ The time it takes for CPE to appear depends on the transfection efficiencyâit 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 5â9.

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<br>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 â20°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 5â10.

11. When &gt;50% of the cells have detached from the plate, prepare viral stock by following Steps 5â9. Name this stock âPrimary Amplificationâ. Store at â20°C.

â¢ 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).<br>â¢ The presence of your recombinant construct can be verified by PCR or Western blotting (Part D) .

12. Determine adenoviral titer (Appendix D). 

The Adeno-Xâ¢ 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.