基于人类腺病毒5型高容量腺病毒载体的克隆及规模化生产

The overall goal of this procedure is to produce a gene-deleted, high-capacity adenoviral vector for delivery and expression of therapeutic transgenes in living cells or organisms. This method can help the gene therapy field to deliver large trans genes that do not fit into other viral vectors. The main advantage of this technique is that high-capacity adenoviruses are devoid of viral genes.

Using these vectors up to 35 kb of foreign DNA can be delivered. The application of this technique extends to our therapy of inherited diseases because it helps to deliver therapeutic ventures for gene correction or gene addition. This method provides an advanced tool for the biodelivery of therapeutic transgenes and can be applied in cell culture as well as living organisms.

Visual demonstration of this method is crucial as vector amplification and vector purification requires some experience. One needs to have right touch during vector purification. Check an aliquot of one in ten diluted linearized high-capacity adenovirus genome expression plasmid by gel electrophresis.

A nine kilobase fragment for the pAd-FTC plasmid backbone and a second, 28 to 36 kilobase DNA fragment for the high-capacity adenovirus genome, plus gene of interest, should be detectable. Next, transfect a 60 milometer dish of one one six cells at 50 to 80%confluency with five micrograms of the linearized high-capacity adenovirus genome, according to standard procedures. 16 to 18 hours post-transfection, carefully remove the medium and add three milliliters of fresh medium.

Then, infect the cells with helper virus AdNG 163R2, applying five transducing units per cell. Incubate in a tissue culture incubator at 37 degrees Celsius and 5%CO2. And agitate the plate every 20 minutes during the first hour of incubation to equally distribute the helper virus to all cells.

48 hours post infection, harvest the cells by flushing them from the culture dish using the culture medium. Reserve a 0.5 milliliter aliquot of the passage zero cells for isolating genomic DNA. Then, repeatedly freeze/thaw a 2.5 milliliter aliquot of passage zero cells by immersing in liquid nitrogen.

Or placing the cells in the negative 80 degrees Celsius freezer and then transferring to a 37 degrees Celsius water bath 3 to 4 times. Mix the 2.5 milliliters of lysate with one milliliter of fresh medium and add helper virus at two transforming units per cell. Aspirate the media from a 60 millimeter dish of one one six cells at 90 to 95%confluency and add the viral mixture to the cells.

Incubate the cells for 48 hours as before. Then, harvest the cells and repeat the freeze/thaw and infection procedures twice to obtain lysates of passage one and passage two. After the passage two lysate has been obtained, mix the 2.5 milliliters of lysate with 17.5 milliliters of fresh media and add two transforming units per cell of helper virus.

Aspirate the media from an 80 to 100%confluent 150 millimeter dish of one one six cells and infect the cells with the viral mixture. 48 hours post infection, harvest the passage three lysate using the procedure just shown. To begin this step, add 900 milliliters of pre-warmed media to a three liter spinner culture flask.

Remove the media from at least 10 individual 150 millimeter dishes of one one six cells at 90 to 100%confluency. And flush off the cells with 10 milliliters of fresh, pre-warmed media. Pipette up and down several times to get a homogeneous cell suspension.

And then transfer the cells directly into the spinner flask. Incubate the spinner flask on a magnetic stirrer in a tissue culture incubator for 24 hours at 37 degrees Celsius and 5%CO2. Adjust the magnetic stirrer to 70 RPM to avoid attachment of cells to the glass surfaces.

24 hours after setting up the spinner culture flask, add 500 milliliters of fresh media. Repeat this step again after a further 24 hours. 72 hours after setting up the culture, add one liter of fresh media to the flask, resulting in a total volume of three liters of cell suspension.

24 hours later, harvest the one one six suspension cells by centrifugation for ten minutes at 500 times g at room temperature. Discard the supernatant and resuspend the pellet in 150 milliliters of media by pipetting up and down about eight to ten times. Transfer the cells to a 250 milliliter storage bottle equipped with a sterile magnetic stir bar.

Then, co-infect the cells with the passage three lysate and two transforming units of helper virus per cell. Here the total cell number is about nine times ten to the eighth cells, therefore, one point eight times ten to the ninth transforming units are added. Stir the cell virus mixture on a magnetic stirrer in the tissue culture incubator for two hours at 60 RPM.

Make sure that the storage bottle is not completely closed to allow air circulation. Two hours post infection, transfer the total volume of the cell virus mixture back into the three liter spinner culture flask. Add 1850 milliliters of fresh, pre-warmed media and incubate in the tissue culture incubator for 48 hours at 70 RPM.

After 48 hours, transfer the cell virus suspension to 500 milliliter centrifuge tubes. And then, centrifuge for 10 minutes at 890 times g at room temperature. Remove the medium and resuspend the pelleted cells in a total volume of 28 milliliters of DPBS.

Store the cell virus suspension at negative 80 degrees Celsius until ready for virus purification. To prepare the viral lysate for cesium chloride gradient purification, freeze/thaw the cell virus suspension four times. Then, centrifuge the viral lysate at 500 times g for eight minutes at room temperature.

Then, collect the supernatant containing the high-capacity adenovirus. Carefully and slowly pipette cesium chloride solutions into the tubes in the following order, 0.5 milliliters of 1.5 grams per cubic centimeter solution, three milliliters of 1.35 grams per cubic centimeter solution and 3.5 milliliters of 1.25 grams per cubic centimeter solution. Overlay approximately 4.5 milliliters of cleared vector supernatant on top of the 1.25 grams per cubic centimeter layer.

Centrifuge the gradients in an ultra centrifuge using a swing out rotor at 12 degrees Celsius for at least two hours at 226, 000 times g to separate the high-capacity adenovirus genome containing viral particles from empty particles and cell debris. Following the centrifugation, a diffuse band of cell debris forms on top of the tubes. Below this, two white bands can be observed.

The upper band contains empty particles, whereas the lower band is the high-capacity adenovirus. Carefully remove the layers of cell debris and empty particles. Then, collect one milliliter of the lower bands from each tube.

Transfer the virus into a sterile 50 milliliter tube. Add up to 24 milliliters of 1.35 grams per cubic centimeter cesium chloride solution to the collected virus particles and mix carefully. Fill the centrifuge tubes to the top with 1.35 grams per cubic centimeter cesium chloride virus solution.

Then centrifuge overnight at 226, 000 times g at 12 degrees Celsius in an ultra centrifuge using a swing out rotor with slow acceleration and deceleration. Remove the upper layers from the top using a pipette. Then, collect the high-capacity adenovirus present in the prominent lower band using a fresh pipette tip.

Finally, dialyse the collected virus particles for buffer exchange. And then, titer the final preparation according to the detailed instructions in the written protocol. This histogram shows absolute numbers of particles for a single vector preparation as determined with three different methods, OD titer measured by optical density, infections titer and helper virus contamination measured by qPCR.

The ratio between the total infectious, high-capacity adenovirus particles and helper virus contamination levels measured by qPCR is shown here. If a fluorescent marker gene is present in your vector, fluorescent microscopy can be performed to monitor the pre-amplification of the vector and to characterize the infectivity of the final vector preparation. After watching this video, you should have a good understanding of how to produce gene deleted, high-capacity adenoviruses.

Especially how to insert your transgene and how to amplify and purify the vectors.