Vergadering en zuivering van Prototype schuimend Virus Intasomes

The overall goal of this procedure is to assemble and purify prototype foamy virus intasomes which may be then be used for biochemical, structural and kinetic studies. This method can help answer key questions in the retrovirology field such as the mechanics and dynamics of integration. The main advantage of this technique is that it allows for the isolation of integration complexes rather than a heterogeneous mixture of species.

Visual demonstration of this method is beneficial as a recovery and solubilization of integration complexes are critical to ensure optimal intasome yield. To begin, combine 1XTEN buffer, 10 micromolar oligo-1 and 10 micromolar oligo-2 in a final volume of 1.5 milliliters. Then, aliquot 25 microliters of the mixture into 60 0.2 milliliter polymerase change reaction tubes.

Anneal the DNA using a thermocycler with the times and temperatures listed in the text protocol. Following the reaction, combine the annealing reactions from all 60 tubes. Then load 500 microliters of two 0.5 milliliter three kilodalton molecular weight cutoff ultracentrifugal filter concentration units.

Concentrate the annealed viral DNA by centrifugation in a microcentrifuge at 14, 000 times times g for 10 minutes at room temperature. After discarding the flow through, add 250 microliters of the remaining annealed viral DNA to each filter unit. Repeat the spin until approximately 50 microliters remain and discard the flow through.

Then, bugger exchange the sample into TE buffer by washing three times with 450 microliters of TE.Following buffer exchange, invert the filter unit and spin at 1000 times g for two minutes at room temperature. The final retented volume for each filtering unit should be approximately 50 microliters. Combine the retentates and transfer to a 1.5 milliliter screw cap tube.

Finally, measure the 260 nanometer ultraviolet absorbence of the viral DNA to calculate DNA concentration. To assemble the intasomes, combine 50 millimolar bis-tris propane pH 7.5, 500 millimolar sodium chloride, 120 micromolar PFV integrase and 50 micromolar viral DNA in a total volume of 150 microliters. Clean a 10 centimeter long piece of 10 millimeter diameter six to eight kilodalton molecular weight cutoff dialysis tubing and the associated clips with double distilled water or the highest purity water available.

Clip one end of the tubing, rinse the inside of the dialysis tubing three times with one to two millimeters of rinse buffer. Remove as much of the rinse buffer as possible with the pipette and thin gel loading tip. Then, transfer the intasome assembly to the dialysis tubing with the pipette and a thin gel loading tip.

Ensure that the assembly is expelled directly into the bottom of the dialysis tubing. Clip the open end of the dialysis tubing minimizing the amount of air introduced within the tubing. Then place the tubing into the dialysis buffer.

Dialyze overnight with gentle stirrings so that the tubing rotates but is not in a vortex. Ensure that the dialysis tubing is submerged and mobile. After approximately one hour, precipitate should be visibile inside the tubing.

Prepare two wide bore pipette tips by removing two to four milliliters from the end of a 200 microliter tip with a new razor or scalpel blade on a clean surface. Remove the dialysis tubing from the dialysis buffer. To prevent dilution of the intasome sample with dialysis buffer that may remain at the end of the tubing near the clip, use a micropipette with a small pipette tip to remove any excess dialysis buffer.

Remove the clip from this end of the dialysis tubing. When recovering the assembly, it is important to collect as much of the solution and precipitate as possible. Use the wide bore propette tip to transfer the sample, including the precipitate, inside the dialysis tubing, to a 1.5 milliliter tube on ice.

Note the total volume of the recovered material. The total volume is typically approximately 140 microliters. The sample with precipitate is at 200 millimolar sodium chloride.

Increase the salt to a final concentration of 320 millimolar sodium chloride by adding the appropriate volume of a stock five molar sodium chloride solution. Transfer the ice bucket with the sample into a four degree Celsius cold room. Use the wide bore pipette tip to re suspend and solubilize the precipitate by pipetting.

Repeat every 20 minutes for at least one hour. Most of the precipitate should go into solution. To purify the intasome, equilibrate a cross-linked, agarose size-exclusion chromatography or SEC column with SEC running buffer at a flow rate of 0.4 milliliters per minute.

Centrifuge the intasome sample in a microfuge at 14, 000 times g for 10 minutes at four degrees Celsius to pellet any remaining precipitate. After carefully removing the supernatant load it onto a 200 microliter injection loop. Apply the sample to the SEC column.

Elute with 25 milliliters of SEC running buffer and collect 95 fractions of 270 microliters. Observe that the SEC chromatogram displays three A280 and A260 peaks. Combine two microliters of each intasome peak fraction in 50 nanograms of three kilobase super coiled plasma DNA in reaction buffer in a final reaction volume of 15 microliters.

Also include a negative control with no added PFV intasome. Incubate at 37 degrees Celsius for five minutes. Stop the reaction with 0.75 microliters of proteinase K and 0.75 microliters of SDS.

Then incubate the sample at 55 degrees Celsius for one hour. Next, add three microliters of 50 percent glycerol to each integration reaction. Load the entire reaction volume to a one percent weight per volume agarose gel.

Also, load one microliter of 10 kilobase DNA size marker to lanes on either side of the samples. In an outer lane, load six microliters of orange G dye. Run the gel at constant voltage, 10 volts per centimeter, at ambient temperature for one hour, or until the orange G dye front reaches the end of the gel.

Immediately image the agarose gel with a fluorescent scanner set to detect ethidium bromide. This image should show concerted integration as a band that runs true to size at three kilobases. After quantifying the bands in each lane with imaging software, select fractions that have the most concerted integration activity.

Distribute each fraction in five microliter aliquots. Finally, snap freeze the aliquots in liquid nitrogen before storing them at minus 80 degrees Celsius. A representative chromatogram of a PFV intasome assembly purification is shown.

Three peak are visible. The center peak correlates to the correctly assembled intasomes. Integration efficiency as a fraction from the center chromatography peak are shown.

Intasomes were tested for activity without and with freeze and thawing. The freezing and thawing showed no difference in the ethidium bromide stained agraose gel of the integration reaction products. Here, super-coiled plasbid, concerted integration products, half sight integration products, and unreacted viral DNA are indicated.

Quantitation of the integration efficiency from the ethidium bromide image also confirms there is no loss of integration activity following freezing and thawing. This allows for long term storage. Label molecule and position affect the efficiency of PFV intasome assembly.

Unlabeled intasomes and an internal Cy5 label display equivalent assembly efficiency. However, end label Cy5 or biotin reduces the assembly efficiency. Once mastered, this technique can be done in two days if it is performed properly.

Following this procedure, other methods like biochemical and single molecule assays can be performed in order to answer additional questions regarding integration, kinetics, and dynamics. After watching this video, you should have a good understanding of how to assemble, purify, and functionally evaluate PFV intasomes.