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The use of viral vectors to introduce exogenous DNA into cells has been in practice since the 1970s (Goff & Berg, 1976). Today, viral vectors are being used for a wide variety of purposes such as:
Viral vectors are capable of doing incredible things, however when they don’t work properly, it can be quite frustrating.
Before beginning your viral transduction, take time to consider what is needed to ensure your experiment is a success! Because of all the different types of target cells, viral vectors, enhancers, and other reagents involved in viral transductions, there are a wide range of conditions you need to optimize in order to achieve the maximum expression levels. We highly recommend using a positive control, such as abm’s Lenti-GFP (Cat. No. LV006) or AAV-GFP, to test different experimental conditions in a multi-well plate to determine optimal conditions for your specific transduction experiment.
In general, here are some tips for a successful transduction:
1. Concentrate and titer your viral stock: Higher viral titers typically increase transduction efficiency! One way to concentrate your virus is to pellet your viral stock by ultracentrifugation and re-suspend into a lower volume. Watch our video on viral titering to get recommendations on different strategies for titering your viral stocks.
2. Optimize your Multiplicity of Infection (MOI): Determine how many viral particles are needed for 100% infection of your target cells. Adding too many viral particles per cell will result in cytotoxicity while adding too few will result in low transduction efficiency. Read our article on how to calculate and determine the optimal MOI for your experiment.
3. Check your virus has been packaged successfully: If your viral construct contains an antibiotic selection marker or a fluorescent reporter, your packaging cells will be selectable under the antibiotic or appear fluorescent under a fluorescent microscope. Watch our video on viral production/packaging to learn more on this topic!
4. Avoid freeze-thaw cycles: When storing or handling your viral stocks, avoid excessive freeze-thawing by aliquoting into smaller stocks or planning your experiment so that you can use your freshly harvested lentiviruses for infection right away.
5. Increase virus-cell contact: You can use transduction enhancers (e.g. polybrene or abm’s ViralEntry™ Transduction Enhancer) to improve viral infectivity. These reagents reduce electrostatic repulsion between the negatively charged cell and viral membranes.
Despite taking all precautions things might still go wrong – not to worry! Our troubleshooting guide will walk you through some common problems as well as our recommendations for how to handle them.
| Problem | Cause | Solution |
| Low Transgene Expression | Transduction efficiency of target cells is too low | Use a transduction enhancer such as abm’s ViralEntry™ Transduction Enhancer (Cat. No. G698) or Polybrene® (Cat. No. G062): These reagents are cationic polymers that reduce the repulsive negative electrostatic forces between the target cells and viral particle membranes (Davis, 2002) to increase virus-cell adsorption. |
| Allow a longer transduction period before harvesting your viruses: Depending on the type of viral vector and target cell used, it may take longer for vectors to enter the cell. For example, Lentiviral vectors have been found to require a minimum of 5 hours to infect target cells (Sevrain, 2016). Before your transduction experiment, test a range of times (e.g. 4-24hrs) to determine optimal conditions for your specific combination of cell and viral vector. | ||
Try a (different) promoter that is optimized for your application:
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| Try a different target cell: primary cells and some suspension cells are known to have lower transduction efficiency while HEK 293, HT1080, HeLa, MDA-MB-468 cells are known to have higher transduction efficiency. | ||
Try a different viral vector:
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| Target cells were not at optimal confluency for transduction | Try a different viral vector: Over-confluent cells will not have sufficient room or nutrients to grow and under-confluent cells may not survive the stress of viral transduction. To determine optimal confluency for your specific target cell/viral vector combination, set up a multi-well plate with a range of confluencies (e.g. 25-50%), and transfect with a reporter-tagged viral vector. Monitor your reporter expression levels to determine optimal confluency needed for maximum expression. | |
| Insufficient time for optimal expression of recombinant protein or selection marker | Allow 72-96 hrs for recombinant protein expression before performing your assay: This ensures there is sufficient accumulation of your desired protein or development of antibiotic resistance, especially for difficult to transduce cell lines. | |
| Viral titer was too low for volume of cells used, volume of media was too high for optimal virus-cell contact | Concentrate your viral stocks: Use ultracentrifugation, filter-based ion exchange chromatography, or size exclusion chromatography to concentrate virus into a smaller volume (Haery, 2016). | |
| Optimize MOI: We recommend performing a pilot experiment using a reporter virus on your target cell line. Simply prepare several transductions with different concentrations of GFP-Virus. Then, use a fluorescence microscope to determine which viral titer yields the highest percentage of infected cells based on GFP expression. Read more about optimizing MOIs here. | ||
| Low Viral Titer | Improper storage (virus ‘dying’) | Avoid freeze/thaw cycles: Store at -80°C and do not thaw unnecessarily. We recommend aliquoting your virus into separate stocks and only thawing an aliquot when you are about to use it. Data provided by our in-house experts shows a 25% loss of viral titer with each freeze-thaw cycle. |
| Add PEG6000 to a final concentration of 5% before freezing down your viral stocks to help stabilize your viruses. | ||
| Insert gene size was too large for vector | Ensure that your gene insert is within the packaging limits of your viral vector (i.e. <8kb for lentivirus, adenovirus, & retrovirus <4.7 kb for AAV). Viral titers will decrease as the insert gene size increases. | |
| Low Target Cell Viability | Cytotoxicity from excessively high MOI (too much virus was used in transduction) | Increase confluency of target cells to slightly higher than 30% at time of transduction. Cells should be no more than 70-80% confluent before transduction. |
| Decrease the amount of virus added: Use smaller volumes or dilute your viral stock. | ||
| Cells were not healthy enough at time of transduction | Monitor target cell health prior to transduction: Ensure cells are contaminant-free (no Mycoplasma) and at least 90% viable. Try our bestselling Mycoplasma PCR Detection Kit which was ranked by a National Health Agency as 6X less expensive while out-performing competitors. | |
| Make sure cells have not been over-passaged (e.g. passage number should be between 3 and 16 for 293 cells), and have not been overgrown before subculturing. | ||
| Gene of interest or enhancer is toxic to cells | Avoid long term exposure of your cells to toxic reagents or gene products: Change growth media 4-24 hrs after transduction or use an inducible expression system. | |
| Use less enhancer reagent: The optimal concentration of Polybrene® depends on cell type (usually 1–8μg/ml) and may need to be empirically determined. | ||
| Try another enhancer such as abm’s ViralEntry™ Transduction Enhancer if your target cells are found to be sensitive to Polybrene® | ||
| Use a different target cell line which is less sensitive to the toxicity of your reagents. |
In addition the troubleshooting tips above, if you are planning on using an Adeno-Associated Virus (AAV), here are some additional factors that could affect viral transduction:
1. Viral Tropism - AAV serotypes
Transduction occurs when a viral vector’s envelope glycoproteins form specific interactions with the cellular receptors on the surface of your target cells, allowing the two to fuse so the vector can enter the cell. The adeno-associated virus (AAV) can be modified to exhibit different capsid surface proteins, called serotypes, to interact with different receptors at the surface of specific cells found in target tissues. Eleven different AAV serotypes have been identified to date, each varying in tropism and therefore making AAV a very useful vector for targeting specific cell types. At abm, we offer serotypes AAV1 through 9, as well as AAVDJ, a hybrid of 8 serotypes with a chimeric capsid of serotypes AAV2, 8, and 9 (Jang, M., 2018), and AAVDJ-8; a mutant of AAVDJ with characteristics of AAV-8 and 9. (Hammond, S. L., 2017). When planning transduction experiments using AAV, it is recommended to select the correct serotype for your target tissue. Use our AAV Serotype Blast™ Kit (AAV099) to determine the optimal serotype for your transduction.
| Target Cell Tissue Type (✓- for recommended application) | |||||
| AAV Serotype | CNS/Retina | Heart | Liver | Lung | Skeletal Muscle |
| AAV1 | ✓ | ✓ | ✓ | ✓ | |
| AAV2 | ✓ | ✓ | ✓ | ||
| AAV3 | ✓ | ✓ | ✓ | ||
| AAV4 | ✓ | ✓ | |||
| AAV5 | ✓ | ✓ | |||
| AAV6 | ✓ | ✓ | ✓ | ✓ | |
| AAV7 | ✓ | ✓ | ✓ | ||
| AAV8 | ✓ | ✓ | ✓ | ||
| AAV9 | ✓ | ✓ | ✓ | ✓ | ✓ |
| AAV DJ | ✓ | ✓ | ✓ | ||
| AAVDJ-8 | ✓ | ✓ | ✓ | ||
2. Gene Insert Size Limit
Another barrier is the size of the gene that can be inserted into the vector. There are two strategies that you can try:
