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How to Optimize your Viral Transduction Experiment: A Troubleshooting Guide

30 min Read

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:

  • Generation of cell lines expressing a tagged or untagged foreign gene
  • Introducing a gene mutation
  • Silencing a gene
  • Vaccine development

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.

5 tips for a successful transduction
Optimizing Viral Transduction 5 tips

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.

Transduction Troubleshooting Guide

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:
  • CMV: yields very strong gene expression in most cells but is prone to silencing in human, and especially mouse or rat cells (alternative: EF1a)
  • EF1a: a strong promoter, highly efficient in stem cells and is good for stable expression in long term cultures
  • PGK: an average promoter that is most proficient in stem cells; sustains stable expression in long term culture of undifferentiated stem cells
  • H1: sustains efficient shRNA, siRNA, and sgRNA transcription for wide range of cell types (RNA Polymerase III promoter) (alternative: U6)
  • CAGGS: a large, strong hybrid promoter (CMV/CBA) with no methylation issues
  • UbC: sustains stable expression but is a relatively low expression promoter
  • MSCV: promotes expression in mouse hematopoietic cells and embryonic stem cells
Use our Lentivirus Promoter Blast™ Kit (LV950) for easy monitoring and optimization of lentiviral transductions with either the CMV, PGK, EF1α, or UBC promoter.
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:
  • AAV: infects both dividing and non-dividing cells with low immunogenicity and pathogenicity, can target a wide range of specific tissue types, and depending on which target cell you use, freeze-thawing them prior to AAV transduction has been found to increase transduction efficiency from 23 to 128-fold by increasing cells’ surface area (Chen et al., 2006)
  • Adenovirus: has a high infection efficiency of dividing and non-dividing cells, stem cells, and primary cells, is non-integrating and has low immunogenicity
  • Lentivirus: has a high infection efficiency of dividing and non-dividing cells, stem cells, and primary cells
  • HSV: delivers large transgenes to a wide variety of mammalian cells, is non-integrating
  • Retrovirus: for the integration of genetic material into dividing cells, inefficient for human cells
  • Baculovirus: generates large amounts of recombinant protein, expresses genes from bacteria, viruses, plants, and mammals
Use our Vector Selection Tool to help you determine which expression system is best for your experiment.
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.
Additional tips for transductions using Adeno-Associated Virus (AAV)

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
AAV DJ    

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:

  • Overlapping vector method: Use two overlapping halves of a transgene coded in two separate rAAV vectors, and simultaneously transduce them into target cells where homologous recombination occurs through the overlapping sequences.
  • Trans-splicing method: Package two non-overlapping halves of a transgene followed by ITR-mediated intermolecular concatemerization and subsequent splicing. This is a more effective method that can result in a 3 to 12-fold increase in the success of producing a full-length transgene product, compared to the previously mentioned overlapping vector method. (Duan et al., 2001)
  • Buchschacher, G. L., Jr, & Wong-Staal, F. (2000). Development of lentiviral vectors for gene therapy for human diseases. Blood, 95(8), 2499–2504.
  • Chen, S., Wasserfall, C., Kapturczak, M. H., Atkinson, M., & Agarwal, A. (2006). Freeze-thaw increases adeno-associated virus transduction of cells. American journal of physiology. Cell physiology, 291(2), C386–C392. https://doi.org/10.1152/ajpcell.00582.2005
  • Davis, H. E., Morgan, J. R., & Yarmush, M. L. (2002). Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent virus adsorption on target cell membranes. Biophysical chemistry, 97(2-3), 159–172. https://doi.org/10.1016/s0301-4622(02)00057-1
  • Duan, D., Yue, Y., & Engelhardt, J. F. (2001). Expanding AAV packaging capacity with trans-splicing or overlapping vectors: a quantitative comparison. Molecular therapy : the journal of the American Society of Gene Therapy, 4(4), 383–391. https://doi.org/10.1006/mthe.2001.0456
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  • Haery, L. 5 Tips for Troubleshooting Viral Transductions. Addgene blog. August 11, 2016, https://blog.addgene.org/5-tips-for-troubleshooting-viral-transductions
  • Hammond, S. L., Leek, A. N., Richman, E. H., & Tjalkens, R. B. (2017). Cellular selectivity of AAV serotypes for gene delivery in neurons and astrocytes by neonatal intracerebroventricular injection. PloS one, 12(12), e0188830. https://doi.org/10.1371/journal.pone.0188830
  • Jang, M., Lee, S. E., & Cho, I. H. (2018). Adeno-Associated Viral Vector Serotype DJ-Mediated Overexpression of N171-82Q-Mutant Huntingtin in the Striatum of Juvenile Mice Is a New Model for Huntington's Disease. Frontiers in cellular neuroscience, 12, 157. https://doi.org/10.3389/fncel.2018.00157
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  • Nieuwenhuis, B., Haenzi, B., Hilton, S., Carnicer-Lombarte, A., Hobo, B., Verhaagen, J., & Fawcett, J. W. (2021). Optimization of adeno-associated viral vector-mediated transduction of the corticospinal tract: comparison of four promoters. Gene therapy, 28(1-2), 56–74.
  • Sevrain, R. 10 best practices for a good transduction efficiency. LentiBlog. August 4, 2016, https://www.vectalys.com/blog/10-best-practices-for-a-good-transduction-efficiency-260
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