Molecular Minutes

A Guide to Lentivirus Production (Protocol, Tips, & more!)

Posted by Applied Biological Materials (abm) on May 1, 2020

Lentivirus-Production_blog-post_main-graphic

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:

  1. Considerations before you start
  2. Basic packaging protocol
  3. Infection test experiment
  4. Storing your lentiviruses
  5. Preventing contamination


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.

 

Recommended: If your lab does not have a Biosafety cabinet, abm offers HEPA Filter Biosafety Cabinets suitable for lentiviral experiments.

 


1. Before you start!

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:

    • Easy to transfect

    • Do not require any specialized media

    • Tend to grow predictably.

    • Contain SV40 Large T-antigen. This allows for episomal replication of transfected plasmids containing the SV40 origin of replication which in turn enables amplification of transfected plasmids and extended temporal expression of the desired gene products. In other words, you will get more viral particles!

2. Determine the titer you need

  • One supernatant collection from transfection will produce a titer of 106 to 107 Infectious Units/ml. You can achieve titers up to 1010 IU/ml by transfecting 293T cells at a larger scale and with additional concentration steps.

 


Lentivirus Production Protocol

 

2. Basic Packaging Protocol

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.

    • If the confluence is too high, there won't be enough room for cells to further divide. If the confluence is too low, there will not be enough viral particles produced.

    • It is important that these cells are healthy and plump, with a fairly even distribution of cells across the plate and no clumps or overcrowding.

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.

    • Lentiviral plasmid: the plasmid vector carrying your gene of interest

    • Packaging plasmids: contains the necessary components for successful viral packaging

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 knowledge base explains the pros and cons of the two systems.


Recommended:abm offers both 2nd generation and 3rd generation packaging systems to suit your needs.

 

Day 3:

  1. Check your dishes for quantifiable transfection efficiency.

    • For example, if the plasmid containing your gene of interest has a fluorescent reporter, you can check for fluorescence.

  2. Perform a complete media change.

    • Be sure to pipette the media onto the side of the plate to avoid disturbing or dislodging the transfected cells.


Recommended: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 4:

  1. Harvest the media from the cells and transfer the supernatant into a sterile bottle or tube. The harvested media contains the lentiviral particles.

  2. Add fresh media back into the plate if you will be performing a second harvest the next day.

  3. Store the supernatant at 4 ̊C if you plan to combine it with a second collection the next day (Day 5).

 

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.

    • Lentiviral particles will still be produced in high quantities until about 72 hours after transfection. After Day 5 viral production becomes negligible.

  2. Collect the supernatant into a sterile bottle or tube.

    • You can combine the second harvest with your first harvest at this time.

  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.


Recommended:If you require a higher titer, commercial kits are available, such as abm’s Speedy Lentivirus Purification system.


Recommended: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!

 


3. Infection Test 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.

    • This can be done by using your lentivirus to infect a standard HEK293T cell line.

  2. Calculate your lentivirus titer.

    • We recommend testing your lentiviral titer, alongside infectivity, so you know what Multiplicity of Infection (MOI) to use in your downstream experiments! Learn more about the importance of MOI here.

 

Recommended:abm’s qPCR Lentivirus Titration Kit provides a simple, one step method of titering your lentiviruses in only 2 hours.

 


4. Storing Your Lentiviruses

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.

 


5. Preventing Contamination

  1. Avoid introducing air bubbles by avoiding techniques such as vortexing.

  2. Make sure you are following the appropriate biosafety guidelines.

  3. Filtering is not recommended, but if you must filter out contaminants, use a low-protein binding 0.45 micron filter or a larger pore-size.

  4. Make sure you are using an appropriate storage container. Don’t store lentiviruses in containers made of materials such as hydrophobic plastics like polystyrene. Instead, use containers and pipette tips made of low protein-binding materials such as polypropylene or silicone.

 

In this post we covered the basic steps for preparing lentiviruses. For detailed step-by-step instructions, download our protocol here.

Topics: Tips and Tricks, Lentivirus, Cell Culture

Molecular Minutes

Educational resources for life scientists and interviews with scientists/science communicators in the field.

For more in-depth articles, check out our knowledge base, which covers topics such as CRISPR, Next Generation Sequencing, PCR, Cell Culture, and more.

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