Frequently Asked Questions:

TransIT® Lentivirus System

Lentivirus production platforms are often limited by insufficient viral titers which require concentration before use. The TransIT® Lentivirus System was developed for enhanced delivery of the essential transfer and packaging vectors required for higher-titer lentivirus production. With the TransIT® Lentivirus System, greater than 2-fold higher functional titers are achieved over competing lentivirus production systems.

GENERAL QUESTIONS AND ANSWERS

Q1. What is lentivirus?
Lentivirus is an enveloped, single-stranded RNA virus from the Retroviridae family commonly used as a gene delivery tool for robust and stable transgene expression in both dividing and non-dividing cells. Stable transgene expression is achieved through the random integration of the viral genome into the host cell genome.

Q2. How is lentivirus produced?
To produce lentivirus, cells are co-transfected with packaging plasmids encoding the required gag, pol, and rev structural and regulatory genes and a transfer vector encoding the gene of interest (GOI). Essential components are combined with the vesicular stomatitis virus (VSV) envelope protein G for a broad virus tropism and increased stability during purification procedures. The lentivirus particles are secreted into the culture medium where they are collected, filtered and frozen into aliquots for subsequent transduction into target cells.

Q3. What components are included in the TransIT® Lentivirus System?
The TransIT® Lentivirus System contains TransIT®-Lenti Transfection Reagent and Lentiviral Packaging Mix Powered by MISSION® Genomics for high-titer lentivirus production in adherent HEK 293T cell types. TransduceIT™ Reagent is also included in the kit for enhanced lentiviral transduction of target cells.

Q4. What is the composition of the TransIT®-Lenti Transfection Reagent?
TransIT®-Lenti Transfection Reagent is a proprietary, animal origin-free formulation composed of a lipid and polymer mixture.

Q5. What vectors/elements are included in the Lentiviral Packaging Mix Powered by MISSION® Genomics?
The Lentiviral Packaging Mix Powered by MISSION® Genomics contains an optimized mixture of the following two plasmids:

pMISSION GAG POL - This packaging vector contains the gag, pol and rev genes required to generate the virion structural proteins and packaging functions. The tat transactivator element is also included for enhanced transcription of the viral genome.
pMISSION VSV-G - The vesicular stomatitis virus (VSV) G-protein vector provides the heterologous envelope for broad virus tropism and increased stability during purification procedures.

These plasmids represent a hybrid second/third generation lentivirus packaging system and are compatible with both second and third generation lentivirus transfer plasmids.

Q6. What is the shelf life of the TransIT® Lentivirus System?
When properly stored and handled, the TransIT®-Lenti Transfection Reagent is guaranteed for 6 months from the date of purchase. The Lentiviral Packaging Mix Powered by MISSION® Genomics and TransduceIT™ Reagent are guaranteed for 1 year from the date of purchase.

Q7. How should the TransIT® Lentivirus System components be stored?
Store the TransIT®-Lenti Reagent, Lentiviral Packaging Mix Powered by MISSION® Genomics and TransduceIT™ Reagent at -20ºC. Before each use, warm to room temperature and vortex gently.

Q8. Can the TransIT®-Lenti Transfection Reagent be used to generate lentivirus in both adherent and suspension HEK 293 cells?
No. TransIT®-Lenti Transfection Reagent is only recommended for adherent cell transfections (e.g. HEK 293T/17) and is not recommended for lentivirus generation in suspension cell types. For highest lentivirus titers in suspension 293 cell types, we recommend using our TransIT-PRO® Transfection Reagent.

Q9. What titers are expected when generating lentivirus with the TransIT® Lentivirus System?
Lentivirus titers vary widely and ultimately depend on the HEK 293T cell subtype, packaging system and transfer vector used for lentivirus generation. Functional titers of ≥ 10⁷ are typically achieved with the TransIT® Lentivirus System when using a MISSION® powered transfer plasmid in HEK 293T/17 cells. For more information, see the TransIT® Lentivirus System product page.

Q10. How does TransIT® Lentivirus System compare to other commercially available lentiviral packaging systems?
Greater than 2-fold higher functional titers are achieved over other commercially available lentiviral production systems when using TransIT® Lentivirus System with a MISSION® powered transfer plasmid. For comparison data, see the TransIT®-Lenti Expression System product page.

PROTOCOL QUESTIONS AND ANSWERS

Q11. Which serum-free medium should I use for preparing TransIT®-Lenti:DNA transfection complexes?
For best results, we recommend using Opti-MEM® I Reduced-Serum Medium for preparing TransIT®-Lenti:DNA complexes. Some serum-free media formulations contain polyanions such as heparin or polydextran sulfate that may inhibit formation of active transfection complexes.

Q12. How much TransIT®-Lenti and total DNA (packaging + transfer plasmid) should be used for transfection?
The quantities of TransIT®-Lenti Reagent and total DNA needed for transfection will depend upon the surface area of the tissue culture well/vessel (see Table 1 in the TransIT®-Lenti Full Protocol). In each well of a 6-well plate, our starting recommendation is 6 µl TransIT®-Lenti Reagent and 2 µg total DNA (1 µg transfer plasmid + 1 µl Packaging Mix) which represents a 3:1 ratio (µl of reagent/µg of DNA, vol:wt).

Q13. What is the recommended transfer-to-packaging plasmid ratio for optimal lentivirus generation?
The TransIT®-Lenti 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.

Q14. 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).

Q15. Is a media change required post-transfection?
No. A media change to remove TransIT®-Lenti:DNA transfection complexes from cells post-transfection is not recommended and may be detrimental to lentivirus titers.

Q16. Are cell density and passage number important factors for successful transfection?
Yes. A cell density of ≥80% confluence at the time of transfection is essential for optimal transfection using TransIT®-Lenti Reagent in cell types typically used for lentivirus generation (e.g. HEK 293T/17). Transfecting at less than 80% confluence may lead to increased cytotoxicity and decreased lentivirus titers. Monitoring passage number is important since 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.

Q17. 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.

Q18. 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®-Lenti 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)

TROUBLESHOOTING QUESTIONS AND ANSWERS

Q19. What can I do to further improve lentivirus titers when using TransIT®-Lenti?
Multiple factors can contribute to lentiviral 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 293T cell types at the time of transfection with TransIT®-Lenti Reagent is ≥80% confluence.
Suboptimal TransIT®-Lenti Reagent to DNA ratio - We recommend using 3 µl TransIT®-Lenti Reagent per 1 µg total DNA (transfer + packaging plasmids).
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 serum-free medium (e.g. Opti-MEM® I Reduced-Serum Medium) and add to cells in complete (serum-containing) growth medium.
Insufficient quantities of DNA for transfection – If using an alternative packaging plasmid system, increasing the amount of total DNA (transfer + packaging plasmids) per reaction while maintaining the recommended 3:1 reagent-to-DNA ratio may help improve titers.

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

Q20. Should I be concerned about cellular toxicity when using TransIT®-Lenti Transfection Reagent?
Toxicity is not typically a concern when over-expressing VSV-G in cultures, as some morphology changes and cell to cell fusion events are expected and do not adversely affect lentivirus titers. However, cellular toxicity 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 is ≥80% 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®-Lenti 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®-Lenti 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.
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:

TransIT® Lentivirus System

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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