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Frequently Asked Questions:

TransIT-VirusGEN® Reagent

The TransIT-VirusGEN® Reagent enables high-titer lentivirus and AAV production in serum-containing adherent cultures, as well as serum-free suspension 293-derived cell types. The TransIT-VirusGEN®, TransIT-VirusGEN® SELECT and TransIT-VirusGEN® GMP Transfection Reagents are identical in formulation, 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 and commercial manufacturing. In addition to the Quality Control testing established for TransIT-VirusGEN® Transfection Reagent, the TransIT-VirusGEN® SELECT Reagent is further qualified with sterility, identity, mycoplasma and endotoxin assays. Building on our commitment to product quality, the TransIT-VirusGEN® GMP Transfection Reagent is manufactured under Current Good Manufacturing Practices (cGMP), which are in compliance with the regulations defined in U.S. 21 CFR 820 from the FDA.

GENERAL QUESTIONS AND ANSWERS

Q1. What is the composition of the TransIT-VirusGEN® Reagent?
Q2. What is the difference between TransIT-VirusGEN® Transfection Reagent, TransIT-VirusGEN® SELECT Transfection Reagent and TransIT-VirusGEN® GMP Transfection Reagent?
Q3. What additional analytical assays are available for the TransIT-VirusGEN® Reagent?
Q4. What configurations are available for TransIT-VirusGEN® Transfection Reagent, TransIT-VirusGEN® SELECT Transfection Reagent and TransIT-VirusGEN® GMP Transfection Reagent?
Q5. What is the shelf life of the TransIT-VirusGEN® Transfection Reagent, TransIT-VirusGEN SELECT Transfection Reagent and TransIT-VirusGEN® GMP Transfection Reagent?
Q6. How should the TransIT-VirusGEN® Reagent be stored?
Q7. Can the TransIT-VirusGEN® Reagent be used to generate lentivirus and AAV in both adherent and suspension HEK 293 cells?
Q8. What titers are expected when generating lentivirus with the TransIT-VirusGEN® Reagent?

PROTOCOL QUESTIONS AND ANSWERS

Q9. Which serum-free medium should I use for preparing TransIT-VirusGEN® Reagent:DNA transfection complexes?
Q10. What is the recommended incubation time for complex formation between TransIT-VirusGEN® Reagent and DNA?
Q11. For lentivirus generation, how much TransIT-VirusGEN® Reagent and total DNA (packaging + transfer plasmid) should be used for transfection?
Q12. What is the recommended transfer-to-packaging plasmid ratio for optimal lentivirus generation?
Q13. For AAV generation, how much TransIT-VirusGEN® Reagent and total DNA (GOI containing transfer + rep/cap + pHelper) should be used for transfection?
Q14. What is the recommended plasmid ratio for optimal AAV generation?
Q15. How should transfections with TransIT-VirusGEN® Reagent be scaled if using culture vessels not listed in the Full Protocol?
Q16. Will antibiotics interfere with my transfection?
Q17. Is a media change required post-transfection?
Q18. Are cell density and passage number important factors for successful transfection?
Q19. At what time point do you recommend harvesting lentivirus post-transfection?
Q20. How can I titer my lentivirus stock?
Q21. At what time point do you recommend harvesting AAV post-transfection?
Q22. How can I titer my AAV stock?

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q23. How can I further improve titers when using TransIT-VirusGEN® Reagent?
Q24. Should I be concerned about cellular toxicity when using TransIT-VirusGEN® Reagent?

GENERAL QUESTIONS AND ANSWERS

Q1. What is the composition of the TransIT-VirusGEN® Reagent?
TransIT-VirusGEN® 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, TransIT-VirusGEN® SELECT Transfection Reagent and TransIT-VirusGEN® GMP Transfection Reagent?
The TransIT-VirusGEN® SELECT Reagent is formulated in the same manner as research-grade TransIT-VirusGEN® Reagent, however additional quality release specifications are performed on the dispensed product for improved compatibility with biopharmaceutical virus manufacture. An even higher standard of quality is applied to the TransIT-VirusGEN® GMP Reagent, which is manufactured under cGMP (following U.S. 21 CFR 820 guidelines) in a dedicated production facility. In addition to the Quality Control testing established for TransIT-VirusGEN® Transfection Reagent, the TransIT-VirusGEN® SELECT and GMP Transfection Reagents are further qualified with sterility, identity, mycoplasma and endotoxin assays.

Q3. What additional analytical assays are available for the TransIT-VirusGEN® Reagent?
Residual reagent and identity tests are available for VirusGEN® products. Please contact techsupport@mirusbio.com for more information.

Q4. What configurations are available for TransIT-VirusGEN® Transfection Reagent, TransIT-VirusGEN® SELECT Transfection Reagent and TransIT-VirusGEN® GMP Transfection Reagent?
TransIT-VirusGEN® Transfection Reagent is available in 0.3 ml (MIR 6703), 0.75 ml (MIR 6704), 1.5 ml (MIR 6700), 5 × 1.5 ml (MIR 6705) and 10 × 1.5 ml (MIR 6706). TransIT-VirusGEN® SELECT Transfection Reagent is available in 30 ml (MIR 6700) and 150 ml (MIR 6735). TransIT-VirusGEN® GMP Transfection Reagent is available in 150 ml (MIR 6845-GMP).

Q5. What is the shelf life of the TransIT-VirusGEN® Transfection Reagent, TransIT-VirusGEN® SELECT Transfection Reagent and TransIT-VirusGEN® GMP Transfection Reagent?
When properly stored and handled, the TransIT-VirusGEN® Transfection Reagent is guaranteed for six months from the date of purchase. The TransIT-VirusGEN® SELECT and GMP Transfection Reagents are guaranteed for a minimum of three months from the date of purchase with recertification of Retest Dates based on real-time stability data. Updated Retest Dates will be revised on the product Certificate of Analysis (CoA). Please contact techsupport@mirusbio.com for the CoA.

Q6. How should the TransIT-VirusGEN® Reagent be stored?
Store the TransIT-VirusGEN® Reagent from -10 to -30ºC. Before each use, warm to room temperature and vortex gently.

Q7. Can the TransIT-VirusGEN® Reagent be used to generate lentivirus and AAV in both adherent and suspension HEK 293 cells?
Yes. TransIT-VirusGEN® 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; Expi293™ Expression Medium for suspension 293 cultures). 

Q8. What titers are expected when generating lentivirus with the TransIT-VirusGEN® 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® Transfection Reagent, TransIT-VirusGEN® SELECT Transfection Reagent and TransIT-VirusGEN® GMP Transfection Reagent product pages.

PROTOCOL QUESTIONS AND ANSWERS

Q9. Which serum-free medium should I use for preparing TransIT-VirusGEN® Reagent:DNA transfection complexes?
For best results, we recommend using Phosphate Buffered Saline (PBS) without calcium or magnesium for preparing TransIT-VirusGEN® 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 and should be avoided. 

Q10. What is the recommended incubation time for complex formation between TransIT-VirusGEN® Reagent and DNA?
Our starting recommendation is 15-60 min. However, users should empirically determine optimal complex formation time for each unique experimental system. Incubating complexes at 4°C may allow for longer, optimal complex formation times.  Importantly, complexes should not be agitated after the initial mixing of reagent and DNA, which may disrupt complexes and result in decreased viral titers.

Q11. For lentivirus generation, how much TransIT-VirusGEN® Reagent and total DNA (packaging + transfer plasmid) should be used for transfection?
The quantities of TransIT-VirusGEN® Reagent and total DNA needed for transfection will depend upon the surface area of the tissue culture well/vessel for adherent 293 cultures or the cell culture volume for suspension 293 cultures (see Table 1 and 2 in the TransIT-VirusGEN® Full Protocol). In each T75 flask, our starting recommendation is 45 µl TransIT-VirusGEN® Reagent and 15 µg total DNA (7.5 µg transfer plasmid + 7.5 µg Packaging Mix) which represents a 3:1 ratio (µl of reagent/µg of DNA, vol:wt).

Q12. What is the recommended transfer-to-packaging plasmid ratio for optimal lentivirus generation?
The TransIT-VirusGEN® 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.

Q13. For AAV generation, how much TransIT-VirusGEN® Reagent and total DNA (GOI containing transfer + rep/cap + pHelper) should be used for transfection?
The quantities of TransIT-VirusGEN® Reagent and total DNA needed for transfection will depend upon the surface area of the tissue culture well/vessel for adherent 293 cultures or the cell culture volume for suspension 293 cultures (see Table 3 and 4 in the TransIT-VirusGEN® Full Protocol). In each T75 flask, our starting recommendation is 45 µl TransIT-VirusGEN® Reagent and 22.5 µg total DNA which represents a 2:1 ratio (µl of reagent/µg of DNA, vol:wt). For a 15 ml suspension culture, our starting recommendation is 45 µl TransIT-VirusGEN® Reagent and 30 µg total DNA which represents a 1.5:1 ratio (µl of reagent/µg of DNA, vol:wt). 

Q14. What is the recommended plasmid ratio for optimal AAV generation? 
The TransIT-VirusGEN® Reagent was optimized using a 1:1:1 weight ratio of pAAV-hrGFP, pAAV-RC and pHelper (AAV Helper-Free System, Agilent Technologies).

Q15. How should transfections with TransIT-VirusGEN® Reagent be scaled if using culture vessels not listed in the Full Protocol?

  • For suspension 293 transfections, use the scaling charts for lentivirus and AAV Production in the TransIT-VirusGEN® Full Protocol to scale per milliliter of total culture.
  • For adherent 293T culture transfections in alternate culture vessels, scale the amounts of TransIT-VirusGEN® 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® Reagent per cm2.
    • For lentivirus transfection scaling: combine + 0.02 ml PBS + 0.2 µg total DNA + 0.6 µl TransIT-VirusGEN® Reagent per cm2.

Q16. 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).

Q17. Is a media change required post-transfection?
No. A media change to remove TransIT-VirusGEN® Reagent:DNA transfection complexes from cells post-transfection is not recommended and may be detrimental to lentivirus and AAV titers.

Q18. 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® 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 × 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.

Q19. At what time point do you recommend harvesting lentivirus post-transfection?
The optimal incubation time for harvesting lentivirus is generally 48 hours post-transfection. Less lentivirus is produced during the period of 48-72 hours post-transfection.

Q20. How can I titer my lentivirus stock?
p24 ELISA assays designed to measure viral-associated p24 capsid protein and qPCR/ddPCR assays 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® 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 transduced 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)

Q21. At what time point do you recommend harvesting AAV post-transfection?
The optimal incubation time for harvesting high-titer AAV is generally 48-72 hours post-transfection but should be determined empirically for each unique experimental system.

Q22. How can I titer my AAV stock?
ELISA assays designed to measure intact AAV particles and qPCR/ddPCR assays 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® Full Protocol describes a method to determine functional titer by transducing HT-1080 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)

NEW Reference Card: Methods to Measure Lentivirus and AAV in Your Sample

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q23. How can I further improve titers when using TransIT-VirusGEN® Reagent?
Mirus Bio has developed Enhancer solutions which further boost AAV and lentivirus titers when used in conjunction with TransIT-VirusGEN® Reagent; the Enhancer solutions are sold together as the VirusGEN® AAV and VirusGEN® LV Transfection Kits. For more information about the VirusGEN® AAV Transfection Kit and VirusGEN® LV Transfection Kit, see the TransIT-VirusGEN® Transfection Reagent, TransIT-VirusGEN® SELECT Transfection Reagent and TransIT-VirusGEN® GMP Transfection Reagent product pages.

Multiple factors can contribute to lentiviral and AAV titers; refer to the following critical troubleshooting tips to improve your transfection results:

  • Optimize cell density (% confluence) at time of transfection - The recommended cell density for adherent 293T cell types at the time of transfection with TransIT-VirusGEN® Reagent is 80-95% confluence. For suspension 293 cells, the recommended density for transfection is 2×106 cells/ml.  
  • Optimize TransIT-VirusGEN® Reagent to DNA ratio - For lentivirus generation, we recommend using 3 µl TransIT-VirusGEN® Reagent per 1 µg total DNA (transfer + packaging plasmids). For AAV generation, we recommend using 1.5-2 µl TransIT-VirusGEN® Reagent per 1 µg total DNA.
  • Use high quality DNA - DNA used for transfection should be highly purified, sterile and free from contaminants such as endotoxin. Remove any traces of endotoxin (e.g. bacterial lipopolysaccharide, LPS) using MiraCLEAN® Endotoxin Removal Kit (MIR 5910 or 5900).
  • Do NOT incubate TransIT-VirusGEN® Reagent without DNA - Diluting TransIT-VirusGEN® Reagent can be performed, but diluted reagent should be mixed with DNA within 5 minutes for optimal transfection complex formation. NOTE: TransIT-VirusGEN® Reagent cannot be filtered after it has been diluted in an aqueous solution such as PBS as this results in loss of critical formulation components
  • Do NOT disturb TransIT-VirusGEN® Reagent:DNA complexes after formation - After the initial mixing of TransIT-VirusGEN® Reagent with DNA, do not agitate or mix the complexes further. Agitation of the complexes will likely result in suboptimal functional virus production.
  • Form complexes in appropriate complex formation solution volume – We recommend forming transfection complexes in 10% of total culture volume, e.g. form complexes in 2.5 ml serum-free medium to transfect 25 ml culture volume. Increasing or decreasing the complex formation solution volume will require optimization of complex formation time.
  • Cells which are normally cultured in serum-containing medium should remain in serum-containing medium during and post-transfection – Though complex formation steps should occur in PBS or serum-free medium (e.g. Opti-MEM®), the cells themselves should be maintained in their normal complete (serum-containing) medium. A cell culture medium change is not necessary.
  • Optimize quantity of DNA used for transfection - Refer to Tables 1-4 in the TransIT-VirusGEN® Full Protocol for recommended total DNA per reaction. Using amounts out of range of 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.

Q24. Should I be concerned about cellular toxicity when using TransIT-VirusGEN® 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® 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® 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.