Editing AAV-Production-293T (section)

= Titration of Recombinant AAV real time PCR.  =

== Extraction of Viral DNA  ==

1. Dilute 2 ÎŒL of the virus stock in 80.5 ÎŒL PCR buffer (50 mM KCl, 10 mM Tris-HCl pH 8.0, 5 mM MgCl2, 0.01% gelatin).<br>2. Add 350 U (17.5 ÎŒL) of DNaseI at 37°C for 30 min.<br>3. Inactivate DNase at 70°C for 10 min.<br>4. Add 10 ÎŒg of proteinase K and incubate 1 h at 50°C.<br>5. Inactivate proteinase K at 95°C for 20 min.<br>7. Store virus DNA at â20°C. 

== Dilution Series of the aav DNA  ==

Transfer 22 ÎŒL vector DNA into a sterile 0.5-mL tube (50X).<br>1. Dilute 2 ÎŒL of the stock solution in step 1 with 20 ÎŒL sterile water (500X).<br>2. Dilute 2 ÎŒL of the stock solution in step 2 with 20 ÎŒL sterile water (5000X).<br>3. Dilute 2 ÎŒL of the stock solution in step 3 with 20 ÎŒL sterile water (50,000X).<br>4. Dilute 2 ÎŒL of the stock solution in step 4 with 20 ÎŒL sterile water (500,000X) and discard 2 ÎŒL of the final dilution.<br>5. Add 80 ÎŒL sterile water to each tube and use 5 ÎŒL per PCR. 

== PCR  ==

Set up standard samples, diluted virus DNA and nontemplate control in duplicates. Add 5 ÎŒL diluted virus DNA, standard, or water per tube, respectively. Then add the following to each tube, so that the total volume for each reaction is 25 ÎŒL. 

WPRE-F (10 uM) 0.5 ÎŒL;<br>WPRE-R (10 ÎŒM) 0.5 ÎŒL;<br>5X SYBR Green Mix 12.5 ÎŒL;<br>H2O 6.5 ÎŒL 

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== Cell Biolab Protocol<br> ==

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1. One day before transfection, plate sufficient 293 cells or 293AAV cells (Cat. # AAV-100) to<br>achieve 70-80% confluence on the day of transfection.<br>2. Cotransfect cells with pAAV Expression vector, pAAV-RC and pHelper.<br>Notes:<br>• We recommend the ratio of vectors at 1:1:1 (pAAV Expression Vector:pAAV-RC:pHelper).<br>• Calcium Phosphate transfection method is preferred for AAV production. For lipid-based<br>transfection reagents, we only suggest FuGENE® 6 (Roche Applied Science) or<br>LipofectamineTM LTX (Invitrogen).<br>3. 48-72 hours after transfection, add 0.5 M EDTA to a final of 10 mM to the plate and incubate for 3<br>min at room temperature. Gently shake the culture plate several times and harvest all media,<br>including cells, in a sterile tube.<br>Notes:<br>• As viral production proceeds, some of the cells will round up and detach from the plate, and<br>can be seen as floating in the medium.<br>• Viruses are present in both intact cells and the growth medium. For more concentrated virus<br>stock, we only recommend proceeding with cell pellet.<br>4. Centrifuge the cell suspension at 1000 RPM for 5 min. Remove the supernatant and resuspend the<br>cell pellet in desired amount of DMEM or sterile PBS.<br><br>

Freeze and thaw the cell suspension four times by placing it alternately in a dry ice/ethanol bath<br>and a water bath of 37oC. Remove cell debris by centrifugation at 10,000 g for 10 min and collect<br>the supernatant as AAV crude lysate.<br>6. AAV crude lysate can be used directly or purified/concentrated if needed. For long term storage,<br>store supernatant at -80oC in aliquots.<br><br>

The quality of rAAV vector preparations is a key element in obtaining reliable and reproducible data.<br>Purification of rAAV from crude cell lysate is usually required before transduction of your target cells.<br>rAAV is usually quantified by genome copy (GC) number. These genome-containing particles are<br>functional vectors that infect target cells. Besides these "full" AAV, empty viral particles are also<br>produced. Measurement of GC rather than total particle number is thus more relevant.<br>1. Concentration and purification of your rAAV: Recombinant AAV vector can be purified by<br>CsCl gradient ultracentrifugation, iodixanol discontinuous gradient ultracentrifugation, and high-<br>performance liquid chromatography (HPLC). For AAV-2, we recommend using Cell Biolabs’<br>ViraBindTM AAV Purification Kit (Catalog # VPK-140).<br>2. Measure titer of your rAAV:<br>a. Genome Copy (GC) Number: This is an important step to ensure consistent viral transduction<br>into your host cell. However, QPCR or dot blot of viral DNA can take as much as 1-4 days to<br>complete. An ELISA method has been developed by using antibody that only reacts with AAV<br>intact particles; however, this method measures all AAV particles including the ones lacking<br>genomic DNA. Cell Biolabs’ QuickTiterTM AAV Quantitation Kit (Catalog # VPK-145) does<br>not involve cell infection; instead it specifically measures the viral nucleic acid content of<br>purified viruses or unpurified viral supernatant sample. The entire procedure takes about 4<br>hours for unpurified supernatant or about 30 minutes for purified AAV.<br>b. Infectious Titer: For AAV vector containing reporter, the rAAV infectious titer can be<br>determined using either green fluorescent protein (GFP) or LacZ as the reporter gene. For<br>rAAV-LacZ, each blue cell after X-Gal staining represents one infectious unit (IU). For rAAV-<br>GFP, each green cell under fluorescence microscopy represents one IU.<br>3. Use transduction reagents to increase infection efficiency: The AAV transduction process<br>includes viral binding and entry, intracellular trafficking, nuclear transport, and viral second strand<br>DNA synthesis. The viral second strand DNA synthesis has been shown to be the rate limiting<br>step, which leads to inefficient transduction by AAV vectors. Cell Biolabs’ ViraDuctinTM AAV<br>Transduction Kit (Catalog # AAV-200) is designed to increase transduction efficiencies by AAV<br>on both dividing and non-dividing cells.<br><br>

[[Category:Protocols]]