Need a gene delivery system that can be used for both in vitro and in vivo studies?
Or a virus that can transduce a broad range of cells with high transduction efficiency?
Look no further than the Recombinant Adenovirus!
In this video, we’ll show you how to clone and package adenoviruses,
create seed stocks, and harvest a high yield!
Recombinant Adenoviruses are wonderful tools for high efficiency gene transfer and
gene therapy due to their ease of production, high transduction efficiency and low immunogenicity.
Once generated, a single adenoviral vector can be used in both in vitro and in vivo applications
and can infect both dividing and non-dividing cells in a broad range of hosts.
Now that's extreme versatility!
Over the past 60 years, scientists have made continuous modifications to the adenovirus genome
to enable it to accommodate larger transgenes and to lower its immunogenicity.
The result is the popular 2nd generation adenoviruses that we will focus on in today's video.
You can learn more about the recombinant adenovirus system at our learning resources center
by clicking on the links in the video description.
Now, let's get started!
In this video we will cover:
I. Adenovirus Cloning and Plasmid Linearization
II. Transfection
III. Virus Amplification
IV. Harvesting and Storing your Adenoviruses
First, you'll need to clone your gene of interest into an adenovirus expression system vector.
There are many commercial cloning kits that can help you speed up this cloning process,
including abm's Adeno-1 to Adeno-8 series of cloning kits that come with various tags and reporters.
Using these kits, you can clone your gene of interest into the provided shuttle vector
at the multiple cloning site in frame with your desired tag or reporter at the C-terminus.
The shuttle vector is utilized because it is easier to manipulate a smaller plasmid.
The expression cassette in pShuttle is flanked by I-CeuI and PI-SceI restriction sites,
which are special homing endonucleases that are unique sites within the large pAdeno vector.
Next, use the I-CeuI and PI-SceI sties to sub-clone your gene of interest, tag or reporter
from the shuttle vector into the pAdeno vector which contains the adenovirus genome required for virus production.
Finally, transform the final ligated recombinant adenovector
into Adeno-Competent cells and select colonies using ampicillin.
Check your recombinant adenovector by restriction digest or sequence verification.
Then amplify and linearize it at the predetermined PacI sties for virus production.
Use the long fragment containing your gene of interest and the adenoviral genome for transfection.
Ensure you have at least 3μg of linearized adenovector for transfection.
Now, we are ready for transfection and virus production.
Seed low passage HEK293 cells in 6-well plates with 2mL of DMEM, 10% FBS, 1% Penicillin-Streptomycin.
Incubate at 37℃, 5% CO2 so that they will be 50-70% confluent at the time of transfection.
Transfection efficiency is highest in actively dividing cells,
therefore, transfection over 70% confluency will negatively impact the cell's ability to uptake DNA.
At room temperature, prepare 2 solutions:
solution A and solution B.
To make solution A, mix 3μg of your linearized adenovector and 100μL serum-free DMEM.
Incubate 5 minutes at room temperature.
For solution B, combine 6μL of abm's DNAfectin Plus transfection reagent with 100μL serum-free DMEM.
Incubate solution B for 5 minutes at room temperature.
Now, combine solutions A and B into a single tube and incubate for 20 minutes at room temperature
to allow formation of DNA-DNAfectin transfection complexes.
This is your transfection solution.
While you're waiting for incubation to complete, aspirate the old growth media from the 6-well plates
and gently wash the cells with fresh serum-free DMEM or sterile PBS
to remove any serum and antibiotics that could interfere with formation of transfection complexes and increase cytotoxicity.
Once incubation is complete, add 800μL of serum-free, antibiotic-free DMEM to the transfection solution.
Next, aspirate the wash solution from the 6-well plates
and add 1mL of the transfection solution to each well.
Label the plates and incubate at 37℃, 5% CO2 for 5 to 8 hours.
After 5 to 8 hours of incubation, aspirate the transfection solution and replace it with 2mL DMEM with 10% FBS and 1% Penicillin-Streptomycin.
If a fluorescent marker was used, you will be able to observe the fluorescent signal 24 hours post-transfection.
Examine your cells under the microscope and note the quality of cells and cytotoxicity levels.
Your transfected cells should reach 80% confluency approximately 2 days after transfection.
Subculture them from the 6-well plate into a 15cm plate with 20mL complete media.
Monitor your cells over the next 7 to 14 days for signs of cytopathicity
characterized by rounded, swollen cells that detach from the plate.
When approximately 90% of the cells have detached form the plate,
harvest the cells and media in a 15mL or 50mL conical tube.
Freeze and thaw the cells three times to ensure complete lysis
by alternating between dry ice and a 37℃ water bath.
Great job!
You have now isolated your adenovirus seed-stock!
Store your seed-stocks at -80℃ until needed.
With your seed-stock, you can easily make high titer virus stocks!
Simply infect a fresh batch of 75% confluent HEK293 cells with the seed-stock
and harvest approximately 3 to 5 days later after cytopathicity has been observed.
In general, up to 2-3 rounds of amplification will yield a titre of 10 pfu/ml from HEK293 supernatant.
This is enough for the transduction of most target cells in vitro at 70-100% efficiency.
Centrifuge the harvested and lysed cells at 3500 rpm for 20 mins
in a benchtop centrifuge to pellet the cell debris.
Transfer the supernatant to a new tube and add 50% glycerol to a final concentration of 5%.
This is your adenovirus stock!
Store your virus stock in a -80℃ freezer.
Excellent! You now have your recombinant adenovirus stock!
To learn more on this topic, visit the link to our blog in the video description.
In our next video, we'll show you how to purify and titrate your adenoviruses
as well as how to infect your target cells!
So, stay tuned and thanks for watching!