Paul.xu at 15:52, 6 October 2011

2011-10-06T15:52:46Z

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== ViraPowerâ¢ Adenoviral Expression System Advantages ==

*Uses Gateway® Technology to allow highly efficient, rapid cloning of a gene of interest into a full-length adenoviral vector, bypassing the need for a shuttle vector and inefficient homologous recombination in human or bacterial cells
*Allows generation of high titer adenoviral stocks (i.e. 1 x 109 pfu/ml in crude preparations and 1 x 1011 pfu/ml in concentrated preparations)
*Efficiently delivers the gene of interest to actively dividing and non-dividing mammalian cells in culture or in vivo
*Generates adenoviral constructs with such a high degree of efficiency and accuracy that the system is amenable for use in high-throughput applications or library transfer procedures
*Allows production of a replication-incompetent virus that enhances the biosafety of the system and its use as a gene delivery vehicle

=== RCA ===

In rare instances, it is possible for homologous recombination to occur between the E1 genomic region in 293 cells and the viral DNA, causing the gene of interest to be replaced with the E1 region, and resulting in generation of a âwild-typeâ, replication-competent adenovirus (RCA) (Lochmuller et al., 1994). This event is most likely to occur during large-scale preparation or amplification of virus, and the growth advantages of the RCA allow it to quickly overtake cultures of recombinant adenovirus. As an alternative, E1-containing producer cell lines such as 911 (Fallaux et al., 1996) or PER.C6 (Fallaux et al., 1998) which contain no regions of homologous overlap with the adenoviral vectors can be used to help reduce the incidence of RCA generation

== Digestion with PacI ==

Before transfecting pAd-DEST expression clone into 293A cells, the plasmid must be digested to expose the left and right viral ITRs to allow proper viral replication and packaging. Each pAd-DEST vector contains Pac I restriction sites (refer to the maps of each vector in the pAd/CMV/V5-DEST and pAd/PL-DEST manual for the location of the Pac I sites). Digestion of the vector with Pac I allows exposure of the left and right viral ITRs and removal of the bacterial sequences (i.e. pUC origin and ampicillin resistance gene). Note: Make sure that your DNA sequence of interest does not contain any Pac I restriction sites.

1. Digest at least 5 ÎŒg of purified plasmid DNA of your pAd-DEST expression construct with Pac I (New England Biolabs, Catalog no. R0547S). Follow the manufacturerâs instructions.

2. Purify the digested plasmid DNA using phenol/chloroform extraction followed by ethanol precipitation or a Qiagen DNA purification kit Note: Gel purification is not required. 3. Resuspend or elute the purified plasmid, as appropriate in sterile water or TE Buffer, pH 8.0 to a final concentration of 0.1-3.0 ÎŒg/ÎŒl.

== Transfection ==

1. The day before transfection, trypsinize and count the 293A cells, plating them at 5 x 105 cells per well in a 6-well plate. Plate cells in 2 ml of normal growth medium containing serum.

2. On the day of transfection, remove the culture medium from the 293A cells and replace with 1.5 ml of normal growth medium containing serum. Do not include antibiotics.

3. Prepare DNA-GeneExpressoMax complexes for each transfection sample by performing the following:

â¢ Dilute 1 ÎŒg of Pac I-digested pAd-DEST expression plasmid DNA in 250 ÎŒl of DMEM without serum. Mix gently.

â¢ Mix GeneExpressoMax gently before use, then dilute 3 ÎŒl in 250 ÎŒl of DMEM medium without serum. Mix gently.

â¢ Combine the diluted DNA with the diluted GeneExpressoMax. Mix gently.

â¢ Incubate for 20 minutes at room temperature to allow the DNA-GeneExpressoMax complexes to form.

4. Add the DNA-GeneExpressoMax complexes dropwise to each well. Mix gently by rocking the plate back and forth. Incubate the cells overnight at 37°C in a CO2 incubator. 5. 48 hours after transfection, remove the medium containing the DNA-GeneExpressoMax complexes and replace with complete culture medium (i.e. D-MEM containing 10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin).

6. 48 hours post-transfection, trypsinize cells and transfer the contents of each well to a sterile 10 cm tissue culture plate containing 10 ml of complete culture medium.

Caution: Remember that you are working with infectious virus at this stage and in all subsequent procedures. Follow the recommended guidelines for working with BL-2 organisms (see page 5 for more information).

7. Replace culture medium with fresh, complete culture medium every 2-3 days until visible regions of cytopathic effect (CPE) are observed (typically 7-10 days post-transfection). For an example, see the next page.

8. Replenish culture medium and allow infections to proceed until approximately 80% CPE is observed (typically 10-13 days post-transfection).

9. Harvest adenovirus-containing cells by squirting cells off the plate with a 10 ml tissue culture pipette. Transfer cells and media to a sterile, 15 ml, capped tube.

== Preparing a Crude Viral Lysate ==

After you have harvested adenovirus-containing cells and media, you will use several freeze/thaw cycles followed by centrifugation to prepare a crude viral lysate. The freeze/thaw cycles cause the cells to lyse and allow release of intracellular viral particles.

1. Place the tube containing harvested cells and media at -80°C for 30 minutes. Remove tube and place in a 37°C water bath for 15 minutes to thaw. Repeat the freezing and thawing steps twice. Note: Do not incubate samples at 37°C for longer than 15 minutes.

2. Centrifuge the cell lysate in a table-top centrifuge at 3000 rpm for 15 minutes at room temperature to pellet the cell debris.

3. Transfer the supernatant containing viral particles to cryovials in 1 ml aliquots. Store the viral stocks at -80°C.

The titer of the initial viral stock obtained from transfecting 293A cells generally ranges from 1 x 107 to 1 x 108 plaque-forming units (pfu)/ml. When stored properly, viral stocks of an appropriate titer should be suitable for use for up to one year.

== Adenovirus Amplification Procedure ==

Follow the procedure below to amplify your adenoviral stock using 293A cells. Make sure that your 293A cells are healthy at the time of plating.

1. The day before infection, trypsinize and count the 293A cells, plating them at 3 x 106 cells per 10 cm plate. Plate cells in 10 ml of normal growth medium containing serum.

2. On the day of infection, verify that the cells are at 80-90% confluency before proceeding. Add the desired amount of crude adenoviral stock (e.g. 100 ÎŒl) to the cells. Swirl the plate gently to mix.

3. Incubate the cells at 37°C in a CO2 incubator and allow infection to proceed until 80-90% of the cells have rounded up and are floating or lightly attached to the tissue culture dish (typically 2-3 days post-infection). This indicates that cells are loaded with adenoviral particles.

Note: If you have used less than 100 ÎŒl of crude viral stock or a lower titer stock for infection, you may need to perform a longer incubation.

4. Harvest adenovirus-containing cells by squirting cells off the plate with a 10 ml tissue culture pipette. Transfer cells and media to a sterile, 15 ml, capped tube.

5. Place the tube containing harvested cells and at -80°C for 30 minutes. Remove tube and place in a 37°C water bath for 15 minutes to thaw. Repeat the freezing and thawing steps twice.

6. Centrifuge the cell lysate in a table-top centrifuge at 3000 rpm for 15 minutes at room temperature to pellet the cell debris.

7. Transfer the supernatant containing viral particles to cryovials in 1 ml aliquots. Store the viral stocks at -80°C.

== Adenoviral Stock Titering ==

1. The day before infection (Day 1), trypsinize and count the cells, plating them such that they will be 80-90% confluent at the time of infection. Incubate cells at 37°C overnight. Example: When using 293A cells, we generally plate 1 x 106 cells per well in a 6-well plate.

2. On the day of infection (Day 2), thaw your adenoviral stock and prepare 10-fold serial dilutions ranging from 10-4 to 10-9. For each dilution, dilute the adenoviral construct into complete culture medium to a final volume of 1 ml. Do not vortex.

3. Remove the culture medium from the cells. Mix each dilution gently by inversion and add to one well of cells (total volume = 1 ml).

4. Swirl the plate gently to disperse the media. Incubate at 37°C overnight.

5. The following day (Day 3), remove the media containing virus and gently overlay the cells with 2 ml of agarose overlay solution per well. Prepare the agarose overlay solution (enough to overlay one 6-well plate at a time) as described below:

â¢ For one 6-well plate (2 ml overlay per well), gently mix 12 ml of prewarmed (at 37°C) plaquing media and 1.2 ml of pre-warmed (at 65°C) 4% agarose. Avoid formation of bubbles.

â¢ Apply the overlay to the cells by gently pipetting the overlay down the side of each aspirated well. Work quickly to prevent premature solidification.

â¢ Place the 6-well plate in a level tissue-culture hood at room temperature for 15 minutes or until the agarose overlay solidifies. Return the plate to a 37°C humidified CO2 incubator.

6. 2 days following the initial overlay (Day 5), gently overlay the cells with an additional 1 ml of agarose overlay solution per well. Prepare the agarose overlay solution as described in Step 5. Allow the agarose overlay to solidify before returning the plate to a 37°C humidified CO2 incubator.

7. Monitor the plates until plaques are visible (generally 8-12 days post-infection = Day 10-14). For each well, gently layer the 5 mg/ml MTT solution (1/10 the volume of the agarose overlay) on top of the solidified agar to stain. Make sure the MTT solution is evenly distributed over the entire surface of the well.

Example: If each well contains 3 ml of agarose overlay, use 300 ÎŒl of 5 mg/ml MTT.

8. Incubate plates for 3 hours at 37°C.

9. Count the plaques and determine the titer of your adenoviral stock.

Vital dye, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Thiazolyl blue (MTT) as a staining reagent to help visualize plaques. Other vital stains including Neutral Red (Sigma, Catalog no. N7005) are suitable. If you wish to use Neutral Red, prepare a 1% solution (100X stock solution) in water and store at +4°C. 

[[Category:Protocols]]

Adenovirus Invitrogen - Revision history

Paul.xu at 15:52, 6 October 2011