The overall goal of this procedure is to produce recombinant parvo viruses, or rec PVS at high titers while preserving their quality and reducing production times and costs. This is accomplished by first producing an initial rec PV viral stock through transfection of HEC 2 93 T cells with rec PV plasmids, together with an adenovirus type five derived vector. Since the introduction of ad genomic elements into the HEC 2 9 3 T cells increases the production of rec PVS by more than tenfold.
In comparison to existing protocols. A novel AD VP helper was generated that contains the genomic adenoviral elements necessary to enhance recombinant parvovirus production as well as the parvovirus VP gene unit. The next step is to produce and purify this novel ADD VP helper according to standard protocols.
The final step in this procedure is to amplify the rec PVS by co infecting NB three 20 4K cells with the rec PV and AD ADP viruses. The efficiency of rec PV production is improved by more than 100 fold with this strategy, which can be used exclusively once a small amount of rec PVS has been produced via transfection. Ultimately, quantitative real-time PCR or a transduction assay is used for the titration of the viral stocks produced.
In addition, a plaque assay is performed to confirm that undesired replication competent viruses have not been generated during the production process. The main advantage of this technique over existing methods like the classical recombinant parvovirus production via plasmid, tion in act and treaty cells is that once we have produced the initial stocks of recombinant parvovirus in adenovirus VP helper, our production will rely entirely on virus infection. T allows the use of NBK cells, which are good producers of parvovirus, but are transfect.
In this way, it is possible to amplify the recombinant per viruses and increase the common power virus heals by more than 100 times reducing production time and cost. Demonstrating the procedure will be Barbara Loic head of the Virus Production Unit would developed with us this novel protocol. We hope that you like this video and we wish you good luck with your experiments.
Production of recombinant parvovirus or rec PV via transfection is required to produce the minimal amount of rec PVS to serve as inoculum in the subsequent production of rec PVS via infection. A biosafety level two laboratory is required for the production of rec PVS one day before transfection seed. 2 million HEC 2 9 3 T cells in a 10 centimeter plate to obtain 50%cellular co fluency.
On the day of transfection grow cells in 10 milliliters of DMEM supplemented with 10%FBS L-glutamine, penicillin and streptomycin incubate overnight at 37 degrees Celsius in 5%carbon dioxide and 92%humidity. On the day of transfection, prepare a virus making mixture in a 1.5 milliliter tube. Add the following reagents in a molar ratio of one to two to one three micrograms of rec PV vector harboring the transgene of interest.
Six micrograms of vector P-C-M-V-V-P harboring the parvovirus capsid gene unit, and eight micrograms of adenovirus derived helper plasmid. Pxx six dilute the plasmid mixture with serum free medium up to 850 microliters to obtain a final concentration of 20 nanograms per microliter. Next, add 42.5 microliters of FU gene HD transfection reagent at a 2.5 to one ratio of FU gene reagent to DNA.
Be careful not to let the FU gene reagent touch the side of the tube vortex gently for five to 10 seconds, and then incubate the transfection mixture at room temperature for 30 minutes. Add the transfection mixture dropwise to the cells. Shake the plate gently to evenly distribute the mixture.
Incubate the plate for 72 hours at 37 degrees Celsius with 5%carbon dioxide and 92%humidity. During this period, progeny parvovirus particles are formed from the parvovirus plasmids to harvest the virus. First, scrape the cells within their medium, then aspirate and transfer the suspension from the plate into a 15 milliliter plastic tube.
Centrifuge the tube at 250 G for five minutes. At room temperature, remove 95%of the supernatant, leaving about 0.5 milliliters behind. Vortex the tube gently to resuspend the pellet into the remaining supernatant and transfer to a 1.5 milliliter einor tube.
Freeze the suspension in a liquid nitrogen bath and then thaw in warm water at 37 degrees Celsius. Vortex the tube vigorously for 15 seconds. Repeat the freeze thaw cycle two more times.
Centrifuge the tube at 16, 000 G for 15 minutes at four degrees Celsius, collect the supernatant into a fresh tube. The supernatant at this stage contains the crude virus extract, which can be used as inoculum for the production of rec PVS via infection. Power virus gain.
A replications depend on cellular factors that are expressed in proliferating cells. Therefore, it is important that the cells used for the recombinant power virus production are in an optimal condition and plated in a proper density as demonstrated in this protocol. One day before infection plate 10 million MB three 20 4K cells in a T 1 75 flask to obtain 60 to 80%cellular co fluency on the following day.
Prepare a second flask containing the same number of cells as a control. Each T 1 75 flask should have 20 milliliters of MEM as a final volume supplemented with 5%FBS L-glutamine, penicillin, and streptomycin on the morning of the day of infection. Prepare a virus mixture in a two milliliter plastic tube.
Add these reagents. ADD VP helper. Add a multiplicity of infection of 10 rec PVS at MOI of 0.5 and MEM to 1.5 milliliters.
Apply the entire virus mixture to one of the T 1 75 flasks. Incubate the flask for two hours at 37 degrees Celsius, 92%humidity and 5%carbon dioxide with gentle shaking every 15 minutes. Incubate for a further 20 to 24 hours at 37 degrees Celsius, 92%humidity and 5%carbon dioxide.
After 20 to 24 hours, replace the culture medium with fresh, medium and incubate the flask under the same conditions for 48 hours as indication of efficient viral production. Clear signs of cytotoxicity should be observed in the flask containing viral infected cells. Starting at 36 to 48 hours post-infection after 48 hours, aspirate and transfer the medium supernatant from the flask into a 50 milliliter centrifuge tube.
Wash the cells twice with 1.5 milliliters PBS trypsin eyes for three to five minutes, and then add the cell suspension to the removed medium centrifuge the cells and the supernatant at 5, 000 G for five minutes at room temperature, discard the supernatant and add one milliliter of TE buffer to the cell pellet. Gently vortex the tube to resuspend the cells. Next, freeze the cell suspension in a liquid nitrogen bath and thaw in warm water at 37 degrees Celsius.
Vortex the tube vigorously for 15 seconds. Repeat the freeze thaw cycle three times. Next, treat the cell suspension with Ben Nase nuclease for 30 minutes at 37 degrees Celsius in order to digest genomic cell and non packaged viral DNAs.
After 30 minutes, centrifuge the tube at 5, 000 G for 20 minutes at four degrees Celsius. Finally collect the supernatant into a fresh tube. The supernatant at this stage contains a crude virus extract.
Typical yields of rec PV GFP produced following this protocol should be in the range of one to 10 times 10 to the eighth viral infectious units. An example of stimulation of rec PV production by an adenovirus based plasmid is shown here. Crude virus extracts containing PHH one GFP erect PV harboring the GFP gene with or without.
The adenovirus based PXX plasmid were applied to NBK indicator cells as observed in these micrographs, there are more GFP positive cells in the presence of PXX six. When GFP transduction assays were performed, a clear increase in rec PV production was obtained in the presence of Pxx six. Production increased from 0.3 GFP transduction units or TU per cell, obtained by conventional protocols to approximately five TU per cell obtained by this protocol.
The results from the GFP transduction assay indicates that the adenovirus E two a, E four, and VA RNA genes contained in PXX six are able to boost parvovirus production. This next figure shows a representative example of rec PV production via infection. NB three 20 4K cells were infected with purified ADP helper virus and then super infected with either of two recombinant parvo viruses.
Crude cell extracts were used to determine virus titers by a transduction assay that showed the AD VP helper further enhanced rec PV production up to greater than 70 T preceded cell. Finally, viral batches produced in the presence or in the absence of ADP were analyzed for their content of replication competent viral particles by plaque assay on MB three 20 4K indicator cells. The results indicate that ADP helper enhanced rec PV production without increasing the occurrence of undesirable replication competent viral particles After its development.
This technique allows scientists in the field of cancer and gene therapy to further upscale the production of recombinant power viruses. Using bioreactors variants can be produced in large amounts needed in a clinical setting.
