A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins

This method can determine both purified and the non-purified AV vac titers and assess the ability for the AAV AAP to promote assembly of cognate and heterologous AV serotype-capsis. The main advantage of this technique is that it is a straightforward inexpensive method that avoids the drawbacks of other AAV titration methods. Though this method has commonly been used for AAV vector quantitation, it can also be applied to address various fundamental questions about AAV biology as shown in this protocol.

On day zero, after culturing HEK 293 cells to 90%confluency, prepare the transfection reagent by mixing the plasmids in 96 microliters of PBS without calcium or magnesium in sterile 1.5 milliliter micro-centrifuge tubes. As indicated in this table, the total amount of DNA is two micrograms per well. Add four microliters of PEI solution to the PBS plasmid DNA mix.

The final volume is approximately 100 microliters or 5%volume of the culture medium. Vortex the tubes briefly and incubate the DNA PEI mixture at room temperature for 15 minutes. Following the incubation, briefly spin the tubes in a micro centrifuge to collect the liquid to the bottom.

And add the DNA PEI mixture drop-wise to the culture medium in each well of the HEK 293 culture plate. Then gently agitate the plates and return them to a 37 degree Celsius incubator with 5%CO2. Transfection of HEK 293 cell is a critical component of this protocol for high-titer virus production.

Healthy, low-passage number cells should be used and care should be taken not to disrupt the cells'monolayer. On days one and two, under an inverted fluorescence microscope, observe cells transfected with the PCMVGFP plasmid to assess the transfection efficiency. On day five, pipette the cells and virus-containing medium up and down or use a cell scraper to scrape all the cells.

Then, transfer the suspension into 15 milliliter polypropylene conical tubes. Store the samples at minus 80 degree Celsius until use. To recover the viral particles, quickly thaw the frozen tubes in a 37 degree Celsius water bath.

Then, vortex the tubes vigorously for one minute to maximize the recovery of AAV from the cells. Pellet the cell debris by centrifugation at 3, 700 times G and four degrees Celsius for 10 minutes. Then transfer 200 microliters of the supernatant from each tube into labeled micro-centrifuge tubes with screw caps for the dot blot assay.

To treat the samples with Serratia marcescens endonuclease, prepare mix A and mix B reagents. Add 10 microliters of each reagent to each sample tube. Vortex the samples for five seconds and briefly spin them to collect the liquid to the bottom of the tubes.

Then incubate the tubes at 37 degrees Celsius for at least one hour. After briefly spinning the samples, carry out proteinase K treatment by preparing mix C reagent and adding 180 microliters to each tube. Then after vortexing and briefly spinning the tubes again, incubate them at 55 degrees Celsius for one hour.

Once the samples have returned to room temperature, add 200 microliters of phenol-chloroform and vortex the tubes for one minute. Then spin the samples in a micro-centrifuge at 16, 100 times G and room temperature for at least five minutes. Transfer 320 microliters of the aqueous layer or 80%of the aqueous fluid volume to a new standard micro-centrifuge tube.

This transfer step is critically important as the same volume must be taken from the sample to maintain accuracy for AAV quantitation. Care should be taken to avoid the organic phase while removing the aqueous layer. Prepare the mix D reagent and add 833 microliters to each sample tube.

After vortexing the tubes, incubate them at minus 80 degrees Celsius for at least 20 minutes. Following centrifugation, pour off supernatant and blot the tubes once on a clean paper towel. Pipette approximately 500 microliters of 70%ethanol into each tube and vortex the tubes for five seconds.

After spinning the samples again, pour off the supernatant and blot the tubes once on a clean paper towel. Then dry the pellets at 65 degrees Celsius before resuspending them in TE buffer. To carry out the dot blot assay, prepare plasmid DNA standards by diluting AAV vectored genome plasmid DNA to 10 nanograms per microliter in water or Tris-HCL buffer.

Transfer a 25 microliter aliquot of the diluted plasmid DNA to a fresh tube and linearize it with an appropriate restriction enzyme in a 50 microliter reaction volume for one hour. Add 450 microliters of water or TE to the tube containing the digested plasmid DNA standard and mix thoroughly. Then transfer 70 microliters of this mixture to a new 1.5 milliliter micro-centrifuge tube with 1, 330 microliters of water or TE to make a diluted plasmid standard.

To set up the dot blot apparatus, using scissors, cut the blotting membrane to an appropriate size for the number of samples and standards. Soak the membrane with water for 10 minutes before placing it on the dot blot apparatus. Then, cover the unused wells.

Add water to the wells to which the samples will be loaded. Apply a vacuum, pull the water through the wells, check for errors, and then retighten the screws. Retest if necessary.

Apply 400 microliters of each diluted plasmid DNA standard to each well and run four lanes of standard dilutions. Use two separate aliquots of the standard digest and load each in duplicate. Then apply 200 microliters per well of each viral DNA sample.

Apply a gentle vacuum to pull the DNA solutions through the wells. Then once all the wells have emptied, release the vacuum by adjusting the three-way valve. Add 400 microliters of one X alkaline solution to each well.

Then wait for five minutes before reapplying the vacuum to empty the wells. Reapply the vacuum and disassemble the dot blot apparatus. Then remove the membrane and rinse it with two X SSC.

Place the membrane on a clean paper towel with the DNA-bound side facing up to remove the excess buffer. Use an appropriate UV crosslinker to UV crosslink the blotted DNA to the membrane. The membrane is now ready for hybridization.

Place the membrane in a hybridization bottle with the DNA-bound side up. Rinse the membrane with five to 10 milliliters of pre-warmed hybridization buffer and discard the buffer. Then add 10 milliliters of pre-warmed hybridization buffer and place the bottle in a rotating hybridization oven set to 65 degrees Celsius.

Rotate the bottle for at least five minutes. Quickly add the denatured sperm DNA and radioactive probe to the hybridization buffer in the bottle and shake it for 10 seconds to mix. Return the bottle to the 65 degree Celsius oven and incubate it with rotation at 65 degrees Celsius for at least four hours.

Once hybridization is complete, stop the rotation. Remove the hybridization bottle and then pour the radioactive probe solution into a 50 milliliter conical tube with a leakproof plug seal cap. To wash the membrane, add 20 to 30 microliters of pre-warmed wash buffer to the hybridization bottle and rotate it for five minutes.

Repeat this wash two more times. After the third wash, remove the membrane from the hybridization bottle, and with paper towels, remove the excess buffer from the membrane. Then place the membrane in a clear plastic paper holder.

With a Geiger counter, check the radioactive signals on the membrane. After exposing the erased phosphor-imaging screen to the membrane, scan the screen using a phosphor-image scanning system and obtain the data on signal intensity of each dot. Carry out data analysis according to the text protocol.

This quantitative dot blot for AAV 2G9 vector titration has two sets of standards and three different amounts of an AAV vector preparation, including 0.3, 0.1, and 0.03 microliters in duplicate. The amounts to be assayed on the blot are determined empirically or based on a preliminary assay. It was determined by interpolation that 0.3, 0.1, and 0.03 microliter aliquots of the AAV 2G9 vector had on average a titer of 5.16, 0.27 nanograms equivalent microliters.

All possible cognate and heterologous serotype combinations of VP3 cas-pid and AAP proteins from AAV5, AAV9, and snake AAV were assessed for capsid assembly by a quantitative dot blot assay. The results of this graph show that AAV5 VP3 assembles regardless of whether AAP was provided in trans. In addition, AAP5 and AAP9 can promote AAV9 VP3 capsid assembly, although AAP5 functions less effectively than AAP9.

Neither AAP5 nor AAP9 exhibits an assembly promoting activity on snake AAV VP3, and snake AAP only promotes assembly of snake AAV VP3. A dot blot analysis was applied to asses nuclease activity of three commercially available nucleases under non-optimized conditions containing cell and culture medium-derived impurities. Enzyme A was found not as active as the other two enzymes, showing that the choice of nuclease could significantly affect the readout of the dot blot assay.

This technique is straightforward for AAV vector quantitation and instrumental in assessing the role of AAP in promoting assembly of AAV capsids derived from cognate and heterologous serotypes. Once mastered, this technique can be done in a day if it performed properly once assay samples are obtained in cell culture experiments. While attempting this procedure, it is important to remember to carefully pipette the correct liquid volume to ensure quantitative accuracy.

After its development, the application of the quantitative dot blot assay to study AAP have paved the way for researchers in the field of AAV biology to further understand the mechanism of AAV capsid assembly. Please do not forget that working with human cells, viral agents, phenol-chloroform, and radioactive materials can be extremely hazardous. And the use of lab coats, gloves, and goggles are needed to limit exposure.

These materials should only be handled in approved areas. After watching this video, you should have a good understanding on how to prepare dot blot samples for AAV quantitation, set up dot blot apparatus, determine AAV titer, and apply the procedure to the study of AAV biology. Following this procedure, other assays like AAV vector transduction assays in culture cells can be performed to answer additional questions, such as the functional titer of vectorized compared to the physical particle titer determined by dot blot assay.