Easy Lentiviral Transduction Protocol

Follow our simple lentiviral transduction protocol and discover expert tips and tricks to make your transductions a success.

Written by: Laura Grassie

last updated: January 23, 2025

Looking for an easy-to-follow lentiviral transduction protocol? This article takes you from experimental design to lentiviral production and confirmation of successful transduction, with helpful hands-on tips to help make your experiment a triumph.

How Lentiviral Delivery Systems Work

The use of viral delivery systems to transduce cells for gene and protein investigations has become prominent over the last 20 years.

One benefit is that lentiviral vectors permit stable expression of your gene of interest. Lentiviruses (a genus of retrovirus) express reverse transcriptase, which converts the viral RNA to double-stranded DNA, and integrase, which inserts this viral DNA into the host DNA.

Once the viral DNA is integrated into the host DNA, it divides along with the host cell, and none are the wiser. The main difference between retrovirus and lentivirus is that retrovirus infects only dividing cells whereas lentivirus infects both dividing and non-dividing cells, [1]  thus making it possible to infect post-mitotic cells like neurons. [2] 

Considerations When Planning Lentiviral Transduction

As we all remember from microbiology class, viruses need cells to “survive” as they lack the replication machinery to produce more copies of their genome. So one of the most important aspects of lentiviral vector delivery system experiments is the actual production of lentiviral vectors, which often takes place in HEK293 cells (or some variety).

For example, one common use of lentivirus delivery systems is to insert short hairpin RNAs (shRNA) for RNAi-mediated gene knockdown. In this instance, the shRNA is first packaged into the lentiviral vector and is then used to transfect HEK293 cells. These transfected cells are allowed to incubate for ~3 days, which permits the lentivirus to replicate and produce lentiviral particles that are then harvested, titered, and used to transduce target cells.

Before starting any experiment, it is always a good idea to plan ahead and order all the required components. For transfection and transduction experiments, you will need:

  • reagents for maxiprep/midiprep;
  • media for growing both HEK293 cells and your cells of interest;
  • transfection reagents like lipofectamine; and
  • the appropriate antibiotic (if using an antibiotic-resistant gene to select transduced cells).

Selection Method

Not all cells will be successfully transduced, and therefore, you will need a way to distinguish or select cells that have been transduced. There are two main ways to achieve this:

  1. Expression of an antibiotic resistance gene. Transduced cells will express the resistance gene, meaning they can survive in the presence of the antibiotic, while non-transduced cells will die.
    This method is a relatively simple way to select your cells of interest.
  2. Expression of a fluorescent marker. For example, your plasmid of interest may also contain a fluorescent protein such as GFP, offering a way to distinguish cells that have been transduced. If your downstream experiments involve microscopy or flow cytometry then this might be a viable alternative to using antibiotics.

Controls for Your Lentiviral Transduction Protocol

Having the appropriate controls is essential to ensure you can rely on your results and is critical for troubleshooting when things go wrong. There are three controls necessary for any lentiviral transduction protocol:

Positive Controls

This allows you to check that the transduction process is successful, so that if your experiment doesn’t work (e.g., you don’t get a knockdown or see exogenous protein expression), you know that the transduction step was not the issue.

A good positive control choice is the transduction of a fluorescent marker. This means you can easily see under a microscope if the transduction step was a success.

Negative Controls

Cells infected with blank lentivirus (as in everything but your DNA of interest) can tell you if the lentiviral transduction step had a negative effect on your cells or help confirm that any phenotype you see is a direct effect of the knockdown or overexpression and not because of non-specific effects of the transduction process or viral infection.

Another important negative control to determine where any phenotypic changes have arisen is untransduced cells. If your experimental cells and blank lentiviral cells are both showing phenotypic changes (e.g., stressed or dying), but your untransduced cells are fine, it’s likely that the transduction is causing issues.

Step-by-step Lentiviral Transduction Protocol

There are 2 main steps to lentiviral transduction:

  1. Generating your packaged virus; and
  2. Transduction of your target cells.

To get good viral titers, you need to first make sure that the cells you will use for viral production are happy and healthy. HEK293 cells are easy to transfect and therefore make an excellent choice for producing your packaged lentivirus.

Keep Your HEK293 Cells Happy

Cells should be routinely checked and split when they reach about 80% confluency.

HEK293 cells are fast-growing and trypsinize quickly, so do not allow them to incubate for long in trypsin (30-60 seconds should do it), or you could witness large cell death.

Cells thawed from frozen stock should be passaged at least twice before seeding for the transduction experiment.

Another important point to remember is to handle dishes containing cells very gently as the cells detach easily.

For the transfection experiment, HEK293 cells should be seeded at about 5 x 106 cells/10 cm dish or in such a way that the cells are about 40-50% confluent the next morning (day of the transfection).

Packaging Up the Virus

Once your cells are ready to transfect, the next step is to package the lentiviral vector plasmid, along with viral packaging and envelope vectors, into liposomes for delivery into HEK293 cells.

It is important to use serum-free media such as OptiMEM for the packaging part of the experiment. Serum-free media enhances the complex formation of DNA with cationic liposomes and thereby increases transfection efficiency.

Reagents for Lentiviral Production:

  • Serum-free media, such as OptiMEM.
  • Lipofectamine, or similar transfection reagent.
  • Plasmid containing DNA of interest (e.g., gene to express or shRNA)
  • Packaging plasmids (3rd Generation use two: one encoding gag and pol, the other encoding rev.
  • Envelope plasmid.
  • HEK293 cells at ~40-50% confluency.
  • Fresh complete media.

Protocol for Lentiviral Production

  1. Add 500 µl of OptiMEM media to two 1.5 ml tubes labeled Tube A and Tube B.
  2. Tube A: add 60 µl of Lipofectamine.
  3. Tube B: Add an equal ratio of the plasmid with shRNA, envelope plasmid, and packaging plasmid to the tube. For example: 10 µg of the shRNA, 5 µg of the plasmid encoding gag and pol, 5ug of the plasmid encoding rev, and 10 µg of the envelope plasmid.
  4. Gently tap the tubes to mix, flash spin, and incubate at room temperature for 10 minutes.
  5. Add contents of Tube B to Tube A dropwise, flick the tube gently to mix, flash spin, and incubate at room temperature for 45 minutes.
  6. At this stage, replace the media on HEK293 cells with 5 ml OptiMEM media.

Now that we have the transfection mixture with the shRNA, packaging plasmids, and lipofectamine, it’s time to start packing.

  1. Tilt the petri dish with HEK293 cells and add the transfection mixture dropwise to the collected medium. At this point, if you are not gentle, you can see the HEK293 cells getting unhappy and dislodging. So please be cautious while adding the transfection mixture.
  2. Carefully swirl the plate to mix up the medium and evenly distribute the transfection mixture onto the cells.
  3. Incubate the cells for 5-8 hours in the serum-free media.
  4. Gently aspirate the media (this will contain any plasmid-containing liposomes that did not transfect the HEK293 cells) and add 10 ml of complete media to the side of the plate so that cells do not detach.

    The HEK293 cells will package the shRNA into a viral particle and release it into the media. From this point onwards, the media will be rich with virus particles, so proper PPE should be worn while handling.
  5. Collect the media (virus particles) after 48 hours and add 10 ml of fresh complete media to the same plate.
  6. Collect the media again after 72 hours and combine it with the media collected at 48 hours.
  7. Filter sterilize the combined collected media by passing it through a 0.45-μm filter to permit virus flow-through.

Freeze-thaw cycles will reduce the potency of the virus, so it is a good idea to make 2.5 ml aliquots of the virus.

The virus can be stored at 4° C for about a month or two, and at -20 °C  long term. Always use bleach to dispose of the pipette tips and dishes used during virus production.

Getting the Virus into the Cells: Protocol for Lentiviral Transduction

Now that we have the virus, it’s time to infect the target cells. In the majority of cases, it is important to titer your virus so that you know the concentration used for your experiment and can reliably repeat your results!

However, in this particular method, we are only aiming to obtain a good knockdown. This is achieved by transducing at least 50% of the cells with the virus.  Use an equal ratio of virus and cells to get 50% transduction.

Reagents for Lentiviral Transduction

  • Cells of interest to transduce.
  • Virus.
  • Polybrene.
  • Fresh complete media.

Protocol for Lentiviral Transduction

  1. In a 15-ml tube, resuspend about 2 million cells (~1/4 of a confluent 10-cm dish) in 2.5 ml complete media.
  2. To the same tube, add 2.5 ml of virus (~1/4 of the total virus obtained from one 10-cm dish), and 15 μg/ml of polybrene.
    Polybrene enhances the transduction efficiency by neutralizing the charges between viral particles and the cell membrane. [3] However, the exact mechanism is not well understood.
  3. Mix gently and plate the cells in a new 10-cm dish.
  4. After 24 hours, replace the media with fresh complete medium without virus.
  5. If the shRNA construct contained a fluorescent marker, you can check the success of the integration 48 hours post-transfection; if not, go ahead with the antibiotic selection for another 48 hours.
    I usually start the transduction on a Wednesday, so the cells can be selected over the weekend.

Confirming Successful Viral Transduction

Successful knockdown or expression of an exogenous protein needs to be confirmed. One simple way to do this is by Western Blotting. For knockdowns, you should see a loss or reduction in your protein of interest.

For exogenous protein expression, you should be able to detect your protein of interest either with an antibody specific to that protein or raised against a tag (e.g., GFP) for tagged proteins.

If you have used a fluorescent marker or are expressing a fluorescently tagged protein, you can check under a microscope or by flow cytometry for the fluorescent signal.

Additional Support For Your Lentiviral Transduction Protocol

We hope that the above lentiviral transduction protocol helps you. But sometimes we all experience issues. Low viral titers can be improved by understanding controlling factors and learning how best to care for your HEK293T cells.

Heard a lot of conflicting information on what you should or shouldn’t do to get good viral production? Separate the fact from the fiction by reading our guide Viral Vector Production: Myths & Misconceptions.

Do we miss any helpful tips? Leave a comment below!

Originally published September 13, 2018. Reviewed and updated April 2022.

References:

  1. Add Gene. Lentiviral Guide. Accessed 21 April 2022.
  2. Matt Carter, Jennifer Shieh, in Guide to Research Techniques in Neuroscience (Second Edition), 2015
  3. Davis, H. E., J. R. Morgan, and M. L. Yarmush. 2002. Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent virus adsorption on target cell membranes. Biophys. Chem. 97:159–172.

Laura holds a PhD in Molecular Biology from the University of Dundee. She has worked several different roles in scientific publishing and now works as a Managing editor at Bitesize Bio.

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