The use of viral delivery systems to transduce cells for gene and protein investigations has become prominent over the last 20 years. In particular, the use of lentiviral vectors permits stable expression of your gene of interest. This is all possible with a little bit of nucleic acid magic.
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 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 cells1, thus making it possible to infect post mitotic cells like neurons2.
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 knock-down. 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 for the study, reagents like lipofectamine for transfection, and the appropriate antibiotic (depending on the antibiotic resistant gene present in your lentiviral vector).
Small RNAs For Targeted Knock-Down
Thanks to the user friendly web applications like siRNA Wizard, BLOCK-iT™ RNAi Designer, shRNA Designer and others, it has now become very easy to design shRNAs. Core facilities in universities may also offer to design and synthesize shRNAs according to your preferences. Always make sure to include a fluorescent marker with the shRNA for easy detection of infected cells. Once the shRNA is obtained, it is a good idea to use midi or maxi-prep, to produce enough for upcoming experiments.
Are Your HEK293 Cells Happy?
It is essential to maintain cells in good condition. 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 for HEK293 cells to incubate for long in trypsin (30-60s 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*10^6 cells/10cm dish or in such a way that the cells are about 40-50% confluent the next morning (day of the transfection).
Packing up for the Transduction
The next step is to package the lentiviral vector plasmid, along with viral packaging 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 there by increases transfection efficiency. For more information please refer here and here!
- Add 500 µl of OptiMEM media to two 1.5 ml tubes labeled Tube A and Tube B.
- Tube A: add 60 µl of Lipofectamine
- Tube B: Add equal ratio of the plasmid with shRNA and packaging plasmid to the tube: 10ug of the shRNA, 5ug of the plasmid encoding gag and pol, and 5ug of the plasmid encoding rev.
- Gently tap mix the tubes, flash spin, and incubate at room temperature for 10 minutes.
- Add contents of Tube B to Tube A drop wise, flick the tube gently, flash spin, and incubate at room temperature for 45 minutes
- At this stage, change the media on HEK293 cells to 5ml OptiMEM media
Now that we have the transfection mixture with the shRNA, packaging plasmids, and lipofectamine, it’s time to start packing.
- Tilt the petri dish with HEK293 cells and add the transfection mixture drop-wise to the collected medium. At this point, if you are not gentle you can see the HEK cells getting unhappy and dislodging. So please be cautious while adding the transfection mixture.
- Once the solution is added, carefully swirl the plate to both mix up the medium and evenly distribute the transfection mixture onto the cells.
Incubate the cells 5-8 hours in the serum free media. After incubation, 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, such 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. Collect the media (virus particles) after 48 hours and add 10 ml fresh complete media to the same plate. Collect media again after 72 hours and combine with the media collected at 48 hours. Filter sterilize the combined collected media by passing it through a 0.45microns filter to permit virus flow-through. Freeze thaw cycles will reduce the potency of the virus, so it is a good idea to make 2.5ml aliquots of the virus. The virus can be stored at 4° C for about a month or two, and at -200C long term. Always use bleach to dispose of the pipette tips and dishes used during virus production. Read and learn a lot more about viral transductions here and here!
Getting the Virus into the Cells
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 virus. We should make sure to maintain the proper ratios to get about 50% transduction :
- Use an equal ratio of virus and cells to get 50% transduction. In a 15ml tube, resuspend about 2 million cells (1/4th of a confluent 10cm dish) in 2.5ml complete media. To the same tube, 2.5ml of virus (1/4th of the total virus obtained from one 10cm dish) and 15ug/ml of polybrene are added. Polybrene3 enhances the transduction efficiency by neutralizing the charges between viral particles and the cell membrane. However, the exact mechanism is not clearly known.
- Mix gently and plate the cells in a new 10cm dish. Wait for 24 hours before changing to fresh complete medium without virus. 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.
You Did It!
Check your cells for knockdown or your specific end point by the appropriate analytical method, typically this would involve a Western Blot.
Please feel free to comment below with any questions. Thanks for reading and good luck!
- Lentiviral Guide, https://www.addgene.org/viral-vectors/lentivirus/lenti-guide/
- Matt Carter, Jennifer Shieh, in Guide to Research Techniques in Neuroscience (Second Edition), 2015
- 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