ViraPower™ T-REx™ Lentiviral Gateway™ Vector Kit - FAQs

In the single-step protocol for the BP/LR Clonase reaction, we would not recommend substituting the BP Clonase II/LR Clonase II enzymes with BP Clonase /LR Clonase enzymes as this would result in very low recombination efficiency.

Yes, we have come up with a single-step protocol for BP/LR Clonase reaction (http://www.thermofisher.com/us/en/home/life-science/cloning/gateway-cloning.html#1), where DNA fragments can be cloned into Destination vectors in a single step reaction, allowing you to save time and money.

We would recommend performing a BP reaction with a Donor vector in order to obtain an entry clone. This entry clone can then be used in an LR reaction with the Destination vector to obtain the new expression clone.

We do not offer the 5X LR Clonase buffer and 5X BP Clonase buffer as standalone products. They are available as part of the enzyme kits.

We do not offer any Gateway vectors for expression in plants.

Possible causes include:

- low transfection efficiency; Use a high-quality plasmid prep, 293FT cells under passage 16, ensure removal of Geneticin during transfection, ensure correct DNA:lipid ratio, and that cells are plated at the correct confluency
- transfected cells are not cultured in medium containing sodium pyruvate; this reagent provides an extra energy source for cells
- viral supernatant harvested too early; viral supernatants can generally be collected 48-72 hrs post-transfection
- viral supernatant too dilute; concentrate virus using CsCl purification
- viral supernatant frozen and thawed multiple times; 3 times should be the maximum freeze/thaw
- gene of interest is large; viral titers decrease as size of insert increases, inserts larger than 5.6 kb are not recommended
- rearrangement in the LTR region of the epxression construct plasmid DNA; use Stb3 cells for transformatin of the lentiviral construct
- poor choice of titering cell line; use HT1080 cells or similar cell line
- Polybrene reagent is not included during transduction; transduce lentiviral construct into cells in the presence of Polybrene reagent
- Lipofectamine reagent handled incorrectly; ensure proper storage and mix gently before use
- Use fluorescence micrscopy to check titer with HiPerform FastTiter lentivirus

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

If 293FT cells detach shortly after transfection (4 hours to overnight):

- This may be a sign of Lipofectamine 2000 toxicity. Cells may have been plated too sparsely prior to transfections.
- The cells may not have been handled gently enough (these cells have a tendency to lift off easily).
- The cells may have been kept at room temperature for too long.

If cells detach 48 to 72 hours post-transfection:
- If the cells lift off in large sheets, this may be a sign of lentivirus production.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

The DNA yield from lentiviral mini-prep DNA is often very low due to the presence of the LTRs in the vector backbone. Hence, we do not recommend using a mini-prep kit for propagation of lentiviral constructs. We recommend preparing lentiviral plasmid DNA using the S.N.A.P. MidiPrep Kit (Cat. No. K191001) or the PureLink HiPure Plasmid Midiprep Kit (Cat. No. K210004), both of which contain 10 mM EDTA in the Resuspension Buffer. Since lentiviral DNA midi-preps also often have low DNA yields, we recommend following specific protocols to increase yield- basically, grow cells slowly, use fewer cells per column, and use 100 mL lentivirus culture for each DNA midi-prep.

Note: If you are going to be mini-prepping the lentiviral plasmid during the cloning/colony screening processes, we recommend using the PureLink HQ Mini Kit (Cat. No. K210001) and following the manual protocol with one change: only a single elution with 50 mL TE, pH 8.0 buffer. The typical yield with this method is normally pretty low, 100-150 ng/mL (i.e., 5-7 mg total). The OD 260/280 is typically between 1.8 and 2.1.

Lentivirus produced using our system is replication-incompetent, and this is a safety feature. You must perform a fresh transfection each time you need more virus.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

The “F” stands for the high transfection efficiency of this particular 293 cell clone (called 293F) and the “T” stands for the SV40 large T antigen. The large T antigen expression plasmid is stably integrated in the genome and confers resistance to Geneticin antibiotic in these cells. The presence of the SV40 large T antigen is important for high-titer lentivirus production and the mechanism is not known. If regular 293 cells or another 293T cell line is used as the producer cell line, you will be able to produce virus, but the titers will be lower.

Our lentiviral packaging mix belongs to the third generation, meaning that it does not express the tat gene. Further, gag/pol and rev genes are supplied as independent plasmids, thus eliminating concerns about recombination events bringing components together as a single vector to produce replication-competent lentivirus.

We carry the Vivid Colors pLenti6.3/V5-GW/EmGFP Expression Control Vector (Cat. No. V37006) and Vivid Colors pLenti6.2-GW/EmGFP Expression Control Vector (Cat. No. V36920), both of which are lentiviral vectors containing Emerald Green Fluorescent Protein (EmGFP). They are designed for use with the ViraPower Lentiviral Expression Systems as positive controls to enable the detection of EmGFP fluorescence following transfection in 293FT cells. These vectors serve as titer controls to produce an EmGFP-expressing lentivirus stock and as a transduction control following transduction in both dividing and non-dividing mammalian cells. Both of these vectors are not cloning vectors. The Vivid Colors pLenti6.3/V5-GW/EmGFP Expression Control Vector has the CMV promoter for driving constitutive expression of EmGFP and the PGK promoter for driving long-term, persistent expression of the blasticidin-stable selection marker, whereas the Vivid Colors pLenti6.2-GW/EmGFP Expression Control Vector has the CMV promoter for driving constitutive expression of EmGFP and the SV40 promoter for driving expression of the blasticidin-stable selection marker. In addition, the Vivid Colors pLenti6.3/V5-GW/EmGFP Expression Control Vector is a HiPerform vector, meaning that it is equipped with two key genetic elements: the Woodchuck Posttranscriptional Regulatory Element (WPRE) and the central Polypurine Tract (cPPT) sequence from the HIV-1 integrase gene to produce at least 4-fold increase in increase in protein expression in most cell types, compared to lentiviral vectors that do not contain these elements.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Yes, the lentiviral expression vector will work as an expression vector by itself and can be stably selected with the appropriate antibiotic. Please note that the vector will be about twice the size of most regular vectors. Therefore, you may need to increase the amount of transfected vector to approximate molar equivalents.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Adenoviral expression is used for transient expression, whereas lentiviral expression is used for longer-term expression. Adenoviral vectors can be amplified several times in 293A cells, whereas the only method to concentrate lentivirus is by centrifugation. Adenovirus requires that host cells have the CAR receptor for efficient transduction, whereas due to the VSVG membrane coat on lentivirus particles, these viruses have broad tropism for a variety of mammalian cell types.

When a co-transfection is performed, there is no way of testing the double stable cell line for functional TetR or GeneSwitch protein, respectively. On the other hand, when sequential transfection is performed, one can functionally test the generated T-REx or GeneSwitch cell line by transiently transfecting the lacZ expression control plasmid and then picking a clone that shows the lowest basal level of expression of lacZ in the absence of the inducer, and the highest level of lacZ in the presence of the inducer. This clone can then be expanded and used to transfect the T-REx or GeneSwitch expression construct, as the case may be.

With the GeneSwitch system, it is possible to have the absolute lowest basal levels of expression of the gene of interest, whereas the T-REx system may be a little leaky due to the inevitable presence of tetracycline in FBS. The induced level of expression in the GeneSwitch system can be even higher than that seen with the CMV promoter. The disadvantage of the GeneSwitch system is that the expression does not appear to switch off very easily in culture, although it has been demonstrated to function beautifully in transgenics. The T-REx system, on the other hand, can be switched on and off by the addition and removal of the inducer.

The Flp-In T-REx system combines the targeted integration offered by the Flp-In system with the powerful inducible expression offered by the T-REx system. It allows generation of isogenic, inducible, stable cell lines and permits polyclonal selection of these cell lines. Once the Flp-In T-REx host cell line containing an integrated FRT site has been created, subsequent generation of Flp-In T-REx cell lines expressing the gene(s) of interest is rapid and efficient.

Doxycycline may be used as an alternative inducing agent in the T-REx system. It is similar to tetracycline in its mechanism of action, and exhibits similar dose-response and induction characteristics as tetracycline in the T-REx system. Doxycycline has been shown to have a longer half-life than tetracycline (48 hours vs. 24 hours, respectively). We do not offer doxycycline, but it may be obtained from Sigma (Cat. No. D9891).

We offer three unique mammalian expression systems for inducible/regulated expression of the gene of interest:

- T-REx system
- Flp-In T-REx system
- GeneSwitch system

Please see below to see how they compare with one another:
System -- Basal Expression Level -- Induced Expression Level -- Response time to Maximal Expression -- Transgenic Appliation
T-Rex system -- Low -- Highest -- High -- Suitable
Flp-In T-REx system -- Lower -- High -- 24-48 hrs -- Suitable
GeneSwitch system -- Lowest -- High -- 24-48 hrs -- Suitable