Lentiviruses are an ideal tool to achieve stable over expression of a gene in your cells of interest.
However, while they are a key tool in today's field of biology
it's common for scientists to be confused on how to produce lentiviruses in their own labs.
Do not fear!
This video will show you how to package and store your lentivirus
so you can begin your gene delivery project.
Before you begin, note that lentiviruses are classified under biosafety level II
as they are able to infect primary human cells.
Ensure your lab meets the requirements of a BSL 2 lab
including a biosafety cabinet to ensure sterility and operator safety.
Before you start packaging your lentivirus
there are a few things you must determine.
1) Your packaging cell line.
The industry standard is 293T cells.
2) What titer you need.
1 supernatant collection will produce a titer of 10^6 to 10^7 IU/ml.
You can achieve higher titers by transfecting cells at a larger scale
with extra concentration steps.
Let's go through the basic steps for packaging your lentivirus.
Day 1
Subculture your cell line for packaging.
Subculture your cells such that they will be 70 to 75% confluence
at the time of transfection.
Cells must be healthy with an even distribution and no clumps.
Day 2
Cotransfect your cell line with the lentiviral plasmids and packaging plasmids.
The lentiviral plasmid carries your gene of interest
while the packaging plasmid provides viral packaging components.
If you are unsure whether you want to use a 2nd or 3rd generation lentiviral packaging system
see our previous video that compares both systems.
Day 3
Check your dishes for quantifiable transfection efficiency.
For example, if your plasmid has a fluorescent reporter
check for fluorescence.
Perform a complete media change.
Pipette the media onto the side of the plate to avoid dislodging transfected cells.
To avoid cells dislodging during media change
we recommend using abm's ProAdhere 293T cells for transfection.
Day 4
Harvest the media from the cells into a sterile bottle or tube and store at 4 ̊C.
The harvested media contains the lentiviral particles.
Make sure your lentivirus storage containers and pipette tips
are made of a low protein binding material like polypropylene or silicone.=
and not made of a hydrophobic plastic like polystyrene.
Add fresh media to the plate if you will perform a second harvest.
Day 5
If you want to collect more viral particles perform a second collection
though viral production will be lower than what you collected on day 4.
Collect the supernatant.
You can combine the second harvest with your first harvest.
Centrifuge the media to pellet any 293T cells inadvertently collected during harvesting.
Filtering is recommended using a 0.45 micron filter to
ensure all cell debris is removed from the viral particles.
Dispose the cells following your institutional biosafety guidelines.
The collected virus may be stored at 4 ̊C for a week
but should be frozen at -80 ̊C for long-term storage.
Avoid freeze/thaw cycles
and consider aliquotting your viral particles into smaller volumes before freezing
so you can can thaw the required amount before use.
abm also offers custom lentivirus packaging services for a range of titers.
We do the necessary lentivirus preparation so you can get right to your experiment.
Congratulations!
You now have a package lentivirus ready for use.
But, wait!
There are two more essential steps before you infect your cells.
1) We recommend performing a small infection test
to test your lentivirus infection performance.
This can be done by using your lentivirus to infect a standard 293T cell line.
2) Calculate your lentivirus titer.
abm’s qPCR Lentivirus Titration Kit provides a simple, one step method
of titering your lentivirus in only 2 hours.
Great work!
You're a lentivirus production champion!
In our upcoming videos, we will cover how to titer and use your lentivirus
to deliver desired genes to your target cells.
To learn about multiplicity of infection
watch our MOI video here.
In the meantime, you can get a copy of the step-by-step protocol from our blog.
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Thanks for watching!