Frequently Asked Questions:

TransIT-VirusGEN® SELECT Transfection Reagent

The TransIT-VirusGEN® SELECT Transfection Reagent enables high titer lentivirus and AAV production in serum-containing adherent cultures, as well as serum-free suspension 293-derived cell types. TransIT-VirusGEN® SELECT is identical in formulation to the research grade TransIT-VirusGEN® Transfection Reagent, which equips researchers with the same reliable and scalable performance and streamlined viral vector production workflow as they progress from discovery and development to clinical trials. In addition to the Quality Control testing established for TransIT-VirusGEN® Transfection Reagent, the TransIT-VirusGEN® SELECT Transfection Reagent is further qualified with sterility, identity, mycoplasma and endotoxin assays.

GENERAL QUESTIONS AND ANSWERS

Q1. What is the composition of the TransIT-VirusGEN® SELECT Reagent?
Q2. What is the difference between TransIT-VirusGEN® Transfection Reagent and TransIT-VirusGEN® SELECT Transfection Reagent?
Q3. What is the shelf life of the TransIT-VirusGEN® SELECT Transfection Reagent?
Q4. How should the TransIT-VirusGEN® SELECT Transfection Reagent be stored?
Q5. Can the TransIT-VirusGEN® SELECT Transfection Reagent be used to generate lentivirus and AAV in both adherent and suspension HEK 293 cells?
Q6. What titers are expected when generating lentivirus with the TransIT-VirusGEN® SELECT Transfection Reagent?

PROTOCOL QUESTIONS AND ANSWERS

Q7. Which serum-free medium should I use for preparing TransIT-VirusGEN® SELECT Reagent:DNA transfection complexes?
Q8. For lentivirus generation, how much TransIT-VirusGEN® SELECT Reagent and total DNA (packaging + transfer plasmid) should be used for transfection?
Q9. What is the recommended transfer-to-packaging plasmid ratio for optimal lentivirus generation?
Q10. For AAV generation, how much TransIT-VirusGEN® SELECT Reagent and total DNA (GOI containing transfer + rep/cap + pHelper) should be used for transfection?
Q11. What is the recommended plasmid ratio for optimal AAV generation?
Q12. How should transfections with TransIT-VirusGEN® SELECT Reagent be scaled if using culture vessels not listed in the Full Protocol?
Q13. Will antibiotics interfere with my transfection?
Q14. Is a media change required post-transfection?
Q15. Are cell density and passage number important factors for successful transfection?
Q16. At what time point do you recommend harvesting lentivirus post-transfection?
Q17. How can I titer my lentivirus stock?
Q18. At what time point do you recommend harvesting AAV post-transfection?
Q19. How can I titer my AAV stock?

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q20. How can I further improve titers when using TransIT-VirusGEN® SELECT Reagent?
Q21. Should I be concerned about cellular toxicity when using TransIT-VirusGEN® SELECT Transfection Reagent?

GENERAL QUESTIONS AND ANSWERS

Q1. What is the composition of the TransIT-VirusGEN® SELECT Reagent?
TransIT-VirusGEN® SELECT Transfection Reagent is a proprietary, animal origin-free formulation composed of a lipid and polymer mixture.

Q2. What is the difference between TransIT-VirusGEN® Transfection Reagent and TransIT-VirusGEN® SELECT Transfection Reagent?
TransIT-VirusGEN® SELECT Transfection Reagent is formulated in the same manner as research-grade TransIT-VirusGEN®, however additional quality release specifications are performed on the dispensed product for improved compatibility with biopharmaceutical virus manufacture. In addition to the Quality Control testing established for TransIT-VirusGEN® Transfection Reagent, the TransIT-VirusGEN® SELECT Transfection Reagent is further qualified with sterility, identity, mycoplasma and endotoxin assays.

Q3. What is the shelf life of the TransIT-VirusGEN® SELECT Transfection Reagent?
When properly stored and handled, the TransIT-VirusGEN® SELECT Transfection Reagent is guaranteed for 6 months from the date of purchase.

Q4. How should the TransIT-VirusGEN® SELECT Transfection Reagent be stored?
Store the TransIT-VirusGEN® SELECT Reagent from -10 to -30ºC. Before each use, warm to room temperature and vortex gently.

Q5. Can the TransIT-VirusGEN® SELECT Transfection Reagent be used to generate lentivirus and AAV in both adherent and suspension HEK 293 cells?
Yes. TransIT-VirusGEN® SELECT Transfection Reagent enables high titer lentivirus and AAV production in both adherent, serum-containing cultures, as well as serum-free suspension 293-derived cell types. For optimal results, culture cells in the appropriate medium per cell type, with or without serum (e.g. DMEM + 10% FBS + 10 mM HEPEs pH 7.4 for adherent 293T cultures; FreeStyle™ F17 + 4 mM L-Glutamine + 0.2% Poloxamer 188 for suspension 293 cultures).

Q6. What titers are expected when generating lentivirus with the TransIT-VirusGEN® SELECT Transfection Reagent?
Virus titers vary widely and ultimately depend on the HEK 293 cell subtype, packaging system and transfer vector used for lentivirus or AAV generation. For more information, see the TransIT-VirusGEN® SELECT Reagent product page.

PROTOCOL QUESTIONS AND ANSWERS

Q7. Which serum-free medium should I use for preparing TransIT-VirusGEN® SELECT Reagent:DNA transfection complexes?
For best results, we recommend using Phosphate Buffered Saline (PBS) without calcium or magnesium for preparing TransIT-VirusGEN® SELECT Reagent:DNA complexes. Serum-free media formulations such as Opti-MEM® I Reduced-Serum Medium can also be used. NOTE: Some serum-free media formulations contain polyanions such as heparin or polydextran sulfate that may inhibit formation of active transfection complexes.

Q8. For lentivirus generation, how much TransIT-VirusGEN® SELECT Reagent and total DNA (packaging + transfer plasmid) should be used for transfection?
The quantities of TransIT-VirusGEN® SELECT Reagent and total DNA needed for transfection will depend upon the surface area of the tissue culture well/vessel (see Table 3 and 4 in the TransIT-VirusGEN® SELECT Full Protocol. In each T75 flask, our starting recommendation is 45 µl TransIT-VirusGEN® SELECT Reagent and 15 µg total DNA (7.5 µg transfer plasmid + 7.5 µl Packaging Mix) which represents a 3:1 ratio (µl of reagent/µg of DNA, vol:wt).

Q9. What is the recommended transfer-to-packaging plasmid ratio for optimal lentivirus generation?
The TransIT-VirusGEN® SELECT Reagent was optimized using a pre-mix of lentivirus packaging vectors. If using individual packaging plasmids, we recommend a starting ratio of 4 µg gag-pol vector, 1 µg rev vector and 1 µg VSV-G vector. Premix the packaging plasmids with an equal amount of the transfer vector (e.g. 6 µg) to maintain a 1:1 (wt:wt) ratio of packaging to transfer plasmids.

Q10. For AAV generation, how much TransIT-VirusGEN® SELECT Reagent and total DNA (GOI containing transfer + rep/cap + pHelper) should be used for transfection?
The quantities of TransIT-VirusGEN® SELECT Reagent and total DNA needed for transfection will depend upon the surface area of the tissue culture well/vessel (see Table 1 and 2 in the TransIT-VirusGEN® SELECT Full Protocol. In each T75 flask, our starting recommendation is 45 µl TransIT-VirusGEN® SELECT Reagent and 22.5 µg total DNA which represents a 2:1 ratio (µl of reagent/µg of DNA, vol:wt).

Q11. What is the recommended plasmid ratio for optimal AAV generation? 
The TransIT-VirusGEN® SELECT Reagent was optimized using a 1:1:1 weight ratio of pAAV-hrGFP, pAAV-RC and pHelper (AAV Helper-Free System, Agilent Technologies).

Q12. How should transfections with TransIT-VirusGEN® SELECT Reagent be scaled if using culture vessels not listed in the Full Protocol?

  • For suspension 293 transfections, use the scaling charts for LV and AAV Production in the TransIT-VirusGEN® SELECT Full Protocol (PDF) to scale milliliter of total culture
  • For adherent 293T culture transfections in alternate culture vessels, scale the amounts of TransIT-VirusGEN® SELECT Reagent, total DNA and serum-free medium for complex formation according to the surface area of the vessel. 
    • For AAV transfection scaling: combine 0.02 ml PBS + 0.3 µg total DNA + 0.6 µl TransIT-VirusGEN® SELECT Reagent per cm2.
    • For lentivirus transfection scaling: combine + 0.02 ml PBS + 0.2 µg total DNA + 0.6 µl TransIT-VirusGEN® SELECT Reagent per cm2.

Q13. Will antibiotics interfere with my transfection?
Some antibiotics are cationic and will inhibit transfection complex formation. We recommend excluding antibiotics from the complex formation step. However, once the transfection complexes are formed, they can be added to cells grown in complete culture medium containing low levels of antibiotics (e.g. 100X stock of penicillin/streptomycin diluted up to 0.1-1X final concentration).

Q14. Is a media change required post-transfection?
No. A media change to remove TransIT-VirusGEN® SELECT Reagent:DNA transfection complexes from cells post-transfection is not recommended and may be detrimental to lentivirus and AAV titers.

Q15. Are cell density and passage number important factors for successful transfection?
Yes. For adherent 293T cell types, a culture density of 80-95% confluence at the time of transfection is essential for optimal transfection using TransIT-VirusGEN® SELECT Reagent. Transfecting at less than 80% confluence may lead to increased cytotoxicity and decreased lentivirus and AAV titers. Monitoring passage number is important as high passage numbers can alter cell characteristics such as morphology and division rate. Maintain HEK 293T/17 cells below passage 30 for optimal recombinant lentivirus production. For suspension 293 cell types, the recommended cell density is 2 x 106 cells/ml. Passage cells 18–24 hours before transfection to ensure that cells are actively dividing and reach the appropriate density at time of transfection.

Q16. At what time point do you recommend harvesting lentivirus post-transfection?
The optimal incubation time for harvesting lentivirus is 48 hours post-transfection. Minimal amounts of functional lentivirus are produced during the period of 48-72 hours post-transfection.

Q17. How can I titer my lentivirus stock?
p24 ELISA assays designed to measure viral-associated p24 capsid protein and qPCR kits designed to amplify a conserved region of the HIV-1 genome (e.g. gag) are common methods for titering lentivirus. However, these methods measure the total amount of viral protein or nucleic acid present which does not equate to functional virus (i.e. virions capable of transducing host cells). The TransIT-VirusGEN® SELECT Transfection Reagent Full Protocol describes a method to determine functional titer by transducing HEK 293T/17 cells with a GFP reporter lentivirus. In this method, cells are tranduced at a low MOI and assayed for GFP expression after 48 hours of incubation. Transducing Units per milliliter (TU/ml) are calculated based on the following formula:

Titer (Transducing Units/ml) = Number of target cells (Count at day 2, transduction) x [% GFP positive cells/100]                                                                                                                    (Volume of Lentivirus Stock in ml)

Q18. At what time point do you recommend harvesting AAV post-transfection?
The optimal incubation time for harvesting high titer AAV is 72 hours post-transfection.

Q19. How can I titer my AAV stock?
ELISA assays designed to measure intact AAV particles and qPCR kits measuring the viral nucleic acid content of purified viruses (genome copy/ml) are commonly used to titer AAV. These methods measure particles or nucleic acid present which does not equate to functional virus (i.e. virions capable of transducing host cells). The TransIT-VirusGEN® SELECT Transfection Reagent Full Protocol describes a method to determine functional titer by transducing HT1080 cells with a GFP reporter AAV. In this method, cells are transduced at a low MOI and assayed for GFP expression after 72 hours of incubation. Transducing Units per milliliter (TU/ml) are calculated based on the following formula:

Titer (HT1080 Transducing Units/ml) = Number of target cells (Count at day 2, transduction) x [% GFP positive cells/100]                                                                                                                   (Volume of AAV Stock in ml)

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q20. How can I further improve titers when using TransIT-VirusGEN® SELECT Reagent?
Multiple factors can contribute to lentiviral and AAV titers; refer to the following critical troubleshooting tips to improve your transfection results:

  • Cell density (% confluence) not optimal at time of transfection - The recommended cell density for adherent 293T cell types at the time of transfection with TransIT-VirusGEN® SELECT Reagent is 80-95% confluence. For suspension 293 cells, the recommended density for transfection is 2 x 106 cells/ml.  
  • Suboptimal TransIT-VirusGEN® SELECT Reagent to DNA ratio -  For lentivirus generation, we recommend using 3 µl TransIT-VirusGEN® SELECT Reagent per 1 µg total DNA (transfer + packaging plasmids). For AAV generation, we recommend using 2 µl TransIT-VirusGEN® SELECT Reagent per 1 µg total DNA.
  • Poor quality of DNA - DNA used for transfection should be highly purified, sterile, and free from contaminants such as endotoxin. Remove any traces of endotoxin (bacterial lipopolysaccharide, LPS) using MiraCLEAN® Endotoxin Removal Kit (MIR 5910 or 5900).
  • Complexes added to cells in serum-free medium – Form complexes in Phosphate Buffered Saline (PBS) without Calcium or Magnesium and add to cells in complete (serum-containing) growth medium. NOTE: Serum-free media formulations such as Opti-MEM® I Reduced-Serum Medium can also be used for transfection complex formation. 
  • Insufficient quantities of DNA for transfection – Refer to Tables 1-4 in the TransIT-VirusGEN® SELECT Full Protocol for recommended total DNA per reaction. Using less than the recommended amounts of total DNA will likely result in suboptimal functional virus production. 

For additional tips, please refer to the Troubleshooting Guide in the user protocol.

Q21. Should I be concerned about cellular toxicity when using TransIT-VirusGEN® SELECT Transfection Reagent?
Morphology changes in HEK 293T cells following transfection with AAV plasmids are expected and indicate virus production. When over-expressing VSV-G in cultures (for lentivirus generation), some morphology changes and cell to cell fusion events are expected and do not adversely affect lentivirus titers. However, cellular toxicity that impacts viral titers may be observed if the transfection protocol is not followed properly. The following are considerations for improving cell health post-transfection:

  • Cell density (% confluence) too low at time of transfection - The recommended cell density at the time of transfection for adherent 293T cell types is 80-95% confluence. If cytotoxicity is observed, increase the cell density to maintain cell health.
  • Plasmid DNA contains high level of endotoxin - DNA used for transfection should be highly purified, sterile, and free from contaminants such as endotoxin. Remove any traces of endotoxin (bacterial lipopolysaccharide, LPS) using MiraCLEAN® Endotoxin Removal Kit (MIR 5910 or 5900).
  • Expressed target gene is toxic to cells - Compare toxicity levels against a cells alone control and cells transfected with an empty vector to assess the cytotoxic effects of the target protein being expressed. If lower levels of target gene expression are desired, consider reducing the amount of target plasmid. Maintain the optimal TransIT-VirusGEN® SELECT Reagent:DNA ratio by using carrier DNA such as an empty cloning vector. For example, use 0.5 µg transfer DNA, 0.5 µg pUC18 and 1 ug packaging plasmids for complexes with a reduced amount of target plasmid.
  • TransIT-VirusGEN® SELECT Reagent:DNA complexes were not mixed thoroughly with the cells in the well – Mix thoroughly to evenly distribute the complexes to all cells. Rock the dish back and forth and side to side to properly distribute the complexes. Do not swirl or rotate the dish, as this may result in uneven distribution. To ensure even complex distribution in larger culture vessels, sterilely remove the complete culture medium from cells and combine with transfection complexes (following the recommended complex incubation) and mix well before adding media + transfection complexes back to the vessel containing cells.
  • Cell morphology has changed – If the passage number of the cells is too high or too low, they can be more sensitive to transfection reagents. Maintain a similar passage number between experiments to ensure reproducibility.
  • Mycoplasma contamination affecting cell health - Check your cells for mycoplasma contamination. Use a freshly thawed stock of cells or use appropriate antibiotics to eliminate mycoplasma.

For additional tips, please refer to the Troubleshooting Guide in the user protocol.

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