Frequently Asked Questions:

VirusGEN® LV Transfection Kit

The VirusGEN® LV Transfection Kit, available in Research Use Only (RUO), SELECT and GMP formats, enables high titer lentivirus production in adherent and suspension 293-derived cell types. The components of the VirusGEN® LV Transfection Kit, VirusGEN® SELECT LV Transfection Kit and VirusGEN® GMP LV Transfection Kit 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. The VirusGEN® SELECT and GMP LV Transfection Kits undergo release testing for sterility, formulation identity, mycoplasma and endotoxin. Building on our commitment to product quality, the VirusGEN® GMP LV Transfection Kit is manufactured under Current Good Manufacturing Practices (cGMP), which follow the regulations defined in U.S. 21 CFR 820 from the FDA.

GENERAL QUESTIONS AND ANSWERS

PROTOCOL QUESTIONS AND ANSWERS

Q12. Which serum-free medium should I use for preparing TransIT-VirusGEN®:DNA transfection complexes?
Q13. Can I filter TransIT-VirusGEN® Reagent?
Q14. How much TransIT-VirusGEN® Reagent and total DNA (transfer + packaging plasmids) should be used for transfection?
Q15. What is the recommended transfer:packaging plasmid ratio for optimal lentivirus generation?
Q16. What is the recommended incubation time for complex formation between TransIT-VirusGEN® Reagent and DNA?
Q17. When should VirusGEN® LV Enhancer be added to the culture?
Q18. Will antibiotics interfere with my transfection?
Q19. Is a media change required post-transfection?
Q20. Are cell density and passage number important factors for successful transfection?
Q21. At what time point do you recommend harvesting lentivirus post-transfection?
Q22. How can I titer my lentivirus stock?

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q23. What can I do to further improve my lentivirus titers when using VirusGEN® LV Transfection Kit?
Q24. Should I be concerned about cellular toxicity when using VirusGEN® LV Transfection Kit?

GENERAL QUESTIONS AND ANSWERS

Q1. What components are included in VirusGEN® LV Transfection Kit?
The VirusGEN® LV Transfection Kit consists of three components: TransIT-VirusGEN® Transfection Reagent, VirusGEN® LV Complex Formation Solution and VirusGEN® LV Enhancer.

Q2. What is the difference between VirusGEN® LV Transfection Kit, VirusGEN® SELECT LV Transfection Kit and VirusGEN® GMP LV Transfection Kit?
All components of VirusGEN® LV Transfection Kits (VirusGEN® LV Transfection Kit, VirusGEN® SELECT LV Transfection Kit and VirusGEN® GMP LV Transfection Kit) are identical in formulation, which makes the transition from R&D to pre-clinical and commercial manufacturing seamless. However, additional quality release specifications are performed for VirusGEN® SELECT and GMP LV Transfection Kits for improved compatibility with biopharmaceutical lentiviral vector manufacture. An even higher standard of quality is applied to VirusGEN® GMP LV Transfection Kit, which is completely manufactured under cGMP (following U.S. 21 CFR 820 guidelines) in a dedicated production facility.

Q3. What analytical assays are available for VirusGEN® LV Transfection Kit?
Residual reagent and identity tests are available for VirusGEN® products. Please contact techsupport@mirusbio.com for more information.

Q4. What configurations are available for VirusGEN® LV Transfection Kit, VirusGEN® SELECT LV Transfection Kit and VirusGEN® GMP LV Transfection Kit?

VirusGEN® LV Transfection Kit (MIR 6760) includes 2×1.5 ml TransIT-VirusGEN® Transfection Reagent, 1×100 ml VirusGEN® LV Complex Formation Solution and 1×100 ml VirusGEN® LV Enhancer.
VirusGEN® SELECT LV Transfection Kit is available in two formats (MIR 6780 and MIR 6785). MIR 6780 includes 1×30 ml TransIT-VirusGEN® SELECT Transfection Reagent, 1×1 L VirusGEN® SELCT LV Complex Formation Solution and 1×1 L VirusGEN® SELECT LV Enhancer. MIR 6785 includes 1×150 ml TransIT-VirusGEN® SELECT Transfection Reagent, 5×1 L VirusGEN® SELECT LV Complex Formation Solution and 5×1 L VirusGEN® SELECT LV Enhancer.
VirusGEN® GMP LV Transfection Kit (MIR 6825-GMP) includes 1×150 ml TransIT-VirusGEN® GMP Transfection Reagent, 5×1 L VirusGEN® GMP LV Complex Formation Solution and 5×1 L VirusGEN® LV Enhancer.

Q5. What is the composition of TransIT-VirusGEN® Reagent?
TransIT-VirusGEN® Reagent is a proprietary, animal origin free formulation composed of a lipid and polymer mixture.

Q6. What is the composition of VirusGEN® LV Complex Formation Solution and VirusGEN® LV Enhancer?
VirusGEN® LV Complex Formation Solution and VirusGEN® LV Enhancer are proprietary, chemically defined and animal origin free formulations.

Q7. What is the mechanism of action of each of the components of VirusGEN® LV Transfection Kit?
TransIT-VirusGEN® Reagent complexes with negatively charged DNA to form lipopolyplexes that associate with the negatively charged cell membrane via electrostatic interactions. These lipopolyplexes are taken into the cell via endocytosis. We believe the enhancer component increases production of certain viral proteins when added between 18-24 hours post-transfection. The enhancer component does not directly impact transfection efficiency because improved lentiviral titers are observed even when the enhancer component is added several hours post-transfection.

Q8. What is the shelf life of VirusGEN® LV Transfection Kit?
When properly stored and handled, VirusGEN® LV Transfection Kit is guaranteed for six months from the date of purchase. VirusGEN® SELECT and GMP LV Transfection Kits 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.

Q9. How should VirusGEN® LV Transfection Kit be stored?
Store TransIT-VirusGEN® Reagent from -10 to -30°C. Store VirusGEN® LV Complex Formation Solution and VirusGEN® LV Enhancer from 4 to 10°C.

Q10. Can VirusGEN® LV Transfection Kit be used to generate lentivirus in both adherent and suspension cells?
Yes. VirusGEN® LV Transfection Kit enables high titer lentivirus 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).

Q11. What titers are expected when using VirusGEN® LV Transfection Kit?
Virus titers vary widely and ultimately depend on the HEK 293 cell subtype, packaging system and gene on the transfer plasmid used for lentivirus generation. For more information, see VirusGEN® LV Transfection Kit product page.

PROTOCOL QUESTIONS AND ANSWERS

Q12. Which serum-free medium should I use for preparing TransIT-VirusGEN®:DNA transfection complexes?
For best results, we recommend using VirusGEN® LV Complex Formation Solution for preparing TransIT-VirusGEN® Reagent:DNA complexes. Phosphate Buffered Saline (PBS) without calcium or magnesium or 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.

Q13. Can I filter TransIT-VirusGEN® Reagent?
TransIT-VirusGEN® Reagent can be filtered with no effect on reagent performance if it has not been diluted in aqueous solution. 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.

Q14. How much TransIT-VirusGEN® Reagent and total DNA (transfer + packaging plasmids) should be used for transfection?
The quantities of TransIT-VirusGEN® Reagent, total DNA, VirusGEN® LV Complex Formation Solution and VirusGEN® LV Enhancer needed for transfection will depend upon cell culture vessel and volume (see Table 1 and 2 in the VirusGEN® LV Transfection Kit 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).

Q15. What is the recommended transfer: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.

Q16. 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.

Q17. When should VirusGEN® LV Enhancer be added to the culture?
VirusGEN® LV Enhancer should be added 18-24 hr post-transfection.

Q18. 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).

Q19. 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 titers.

Q20. 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 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

Q21. 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.

Q22. 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 VirusGEN® LV Transfection Kit 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)

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

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q23. What can I do to further improve my lentivirus titers when using VirusGEN® LV Transfection Kit?
Multiple factors can contribute to lentivirus titers; refer to the following critical troubleshooting tips to improve your transfection results:

Optimize cell density (% confluence) at time of transfection - The recommended confluency at the time of transfection for adherent 293T cell types 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).
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 VirusGEN® LV Complex Formation Solution (or PBS) 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 serum-free medium (e.g. VirusGEN® LV Complex Formation Solution or PBS), 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 Table 1 and 2 in the VirusGEN® LV Transfection Kit 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 VirusGEN® LV Transfection Kit?
When generating lentivirus, overexpression of the vesicular stomatitis virus (VSV) G protein causes changes in cell morphology and can even result in cell-cell fusion. VirusGEN® LV Enhancer may also decrease cell growth. This is normal and does not adversely affect virus 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 confluency at the time of transfection for adherent 293T cell types is 80-95% confluence. For suspension 293 cells, the recommended density for transfection is 2×10⁶ cells/ml. 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 dishes, 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.

Frequently Asked Questions:

VirusGEN® LV Transfection Kit

GENERAL QUESTIONS AND ANSWERS

PROTOCOL QUESTIONS AND ANSWERS

TROUBLESHOOTING QUESTIONS AND ANSWERS

GENERAL QUESTIONS AND ANSWERS

PROTOCOL QUESTIONS AND ANSWERS

TROUBLESHOOTING QUESTIONS AND ANSWERS

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