Lentiviruses are a key tool in today’s field of biology as they provide a reliable way to achieve stable over-expression of a gene in your cells of interest. However, many scientists are confused about how to produce these genetic engineering marvels in their own labs.
In this blog post, we'll outline the steps for packaging lentiviruses, including:
Before you begin, it's important to note that Lentiviruses are classified under Biosafety Level II since they are able to infect primary human cells. Ensure that your lab meets all the requirements of a BSL-II lab before you begin. This includes having a Biolsafety Cabinet to ensure sterility and to protect the operator from the viruses.
If your lab does not have a Biosafety cabinet, abm offers HEPA Filter Biosafety Cabinets suitable for lentiviral experiments. |
Before you start packaging your lentivirus, there are a few things you need to determine:
1. Your packaging cell line
Make sure you use a good packaging cell line to produce the maximum viral titer. The industry standard is to use 293T cells. This is because 293T cells are:
2. Determine the titer you need
Day 1
1. 24 hours before transfection, subculture your 293T cells for packaging in such a way that they will be 70 - 75% confluence at the time of transfection.
Day 2
1. Check and ensure that your cells are healthy, and at the right confluency.
2. Transfect your 293T cells with the lentiviral plasmids and packaging plasmids.
You will need to co-transfect these plasmids into 293T cells to make the lentivirus.
If you are unsure whether you want to use a 2nd or 3rd generation lentiviral packaging system, our introduction to the lentivirus system knowledge base article explains the pros and cons of the two systems.
abm offers both 2nd generation and 3rd generation packaging systems to suit your needs. |
Day 3
1. Check your dishes for quantifiable transfection efficiency.
2. Perform a complete media change.
Use abm’s ProAdhere 293T cells for large-scale transfection as their high adherence to tissue culture-treated plastic substrates prevent dislodging of cells during media changes. |
Day 5
1. If you want to collect more lentiviral particles, you can perform a second collection on this day. However, the viral production will be less than what you collected on Day 4.
2. Collect the supernatant into a sterile bottle or tube.
3. Centrifuge the media to pellet any 293T cells that were inadvertently collected during the harvesting.
4. Dispose the cells safely following your institutional biosafety guidelines.
Viral particles harvested directly from cell supernatant are typically at a titer of 106 IU/ml to 107 IU/ml.
If you require a higher titer, commercial kits are available, such as abm’s Speedy Lentivirus Purification system. |
abm also offers custom lentivirus packaging services for a range of titers. We will do all the necessary preparation of lentiviruses so you can get right into your experiment! |
There are two more essential steps to cover before you move on to infecting your cells.
1. We recommend that you test your lentivirus infection performance in a small experiment.
2. Calculate your lentivirus titer.
abm’s qPCR Lentivirus Titration Kit provides a simple, one step method of titering your lentiviruses in only 2 hours. |
Lentiviral particles can be stored for up to a week at 4°C. However, for long-term storage, samples should be frozen as soon as possible at -80°C. Frozen lentiviruses are stable for at least one year.
One important thing to note is that freeze-thaw cycles can decrease the titer drastically (sometimes as much as 10-fold!). This means you should aliquot your lentiviruses into working volumes before you freeze them.
In this post we covered the basic steps for preparing lentiviruses. For detailed step-by-step instructions, download our protocol here.