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We strongly recommend using Stbl3™ E. coli for cloning lentiviral constructs. Stbl3™ E. coli cells contain the recA13 mutation in their genotype that helps to minimize the likelihood of unwanted recombination between the LTRs. After transforming into Stbl3™ E. coli, we recommend picking colonies and validating the lentivirus DNA from mini-preps using Afl II and Xho I digests before proceeding to midi-preps. In all of our lentiviral vectors, Afl II sites are present in both 5´ and 3´ LTRs, and a Xho I site is present after the 3´ end of the MCS. Assuming Afl II cuts only in the LTR sites, and there are no Afl II or Xho I sites in the insert, 3 DNA fragments are expected to be generated from the Afl II + Xho I digest. Any unexpected DNA fragments can be assumed to be a result of LTR recombination. Only clones with the expected pattern of DNA fragments should be chosen for the subsequent midi-prep.

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.

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.

Here are possible causes and solutions:

Cause

Solution

Low transfection efficiency:

  • Used poor-quality expression construct plasmid DNA (i.e.,plasmid DNA from a mini-prep)
  • Unhealthy 293FT cells; cells exhibit low viability
  • Cells transfected in medium containing antibiotics (i.e.,Geneticin®)
  • Plasmid DNA:transfection reagent ratio incorrect
  • 293FT cells plated too sparsely

 

  • Do not use mini-prep plasmid DNA for transfection. Use the PureLink® HiPure Plasmid Midiprep Kit or CsCl gradient centrifugation to prepare plasmid DNA.
  • Use healthy 293FT cells under passage 16; do not overgrow cells. Culture cells for at least 3–4 passages before transfection.
  • Although Geneticin® is required for stable maintenance of 293FT cells, do not add Geneticin® to medium during transfection, as this reduces transfection efficiency and causes cell death.
  • Use a DNA:Lipofectamine® 2000 ratio ranging from 1:2 to 1:3 (in μg:μL).
  • Plate cells such that they are 90–95% confluent at the time of transfection or use the Reverse Transfection protocol (i.e.,add cells to medium containing DNA-lipid complexes).

Transfected cells not cultured in medium containing sodium pyruvate

One day after transfection, remove medium containing DNA-lipid complexes and replace with medium containing sodium pyruvate. Sodium pyruvate provides an extra energy source for the cells.

Viral supernatant harvested too early

Viral supernatants can generally be collected 48–72 hours post transfection. If many cells are still attached to the plate and look healthy at this point, wait an additional 24 hours before harvesting the viral supernatant. Harvest no later than 72 hours post-transfection.

Viral supernatant too dilute

Concentrate virus using CsCl purification or any method of choice.

Viral supernatant frozen and thawed multiple times

Do not freeze/thaw viral supernatant more than 3 times.

Gene of interest is large

Viral titers generally decrease as the size of the insert increases; inserts larger than 5.6 kb are not recommended.

Gene of interest is toxic to cells

Generation of constructs containing activated oncogenes or potentially harmful genes is not recommended.

Rearrangement in the LTR regions of the expression construct plasmid DNA

  • Make sure to use Stbl3™ cells for transformation of the lentiviral construct
  • Perform restriction digestion of the lentiviral construct plasmid DNA using a combination of Afl II and Xho I enzymes. Afl II sites are present in both LTRs. The Xho I site is present in the plasmid backbone at the 3’ end of the insert. Assuming there are no Afl II or Xho I sites in the insert, 3 DNA fragments are generated from the Afl II + Xho I digest. Any unexpected DNA fragments are a result of LTR recombination.

Poor choice of titering cell line

Use HT1080 cells or another adherent cell line with the characteristics discussed in the manual.

Polybrene® reagent not included during transduction

Transduce the lentiviral construct into cells in the presence of Polybrene® reagent.

Lipofectamine® 2000 handled incorrectly

  • Store at 4°C. Do not freeze.
  • Mix gently by inversion before use. Do not vortex.

Using fluorescence microscopy to check titer with HiPerform™ FastTiter™ lentivirus

With a HiPerform™ FastTiter™ lentiviral vector, signal level of EmGFP in the cells is not optimal for visual evaluation using fluorescence microscopy. We recommend using only flow cytometry to evaluate transduction efficiency.

Here are possible causes and solutions:

Cause

Solution

Too much antibiotic used for selection

Determine the antibiotic sensitivity of your cell
line by performing a kill curve experiment, and use the minimum concentration required to kill your untransduced cell line.

Viral stocks stored incorrectly

Aliquot and store stocks in cryovials at –80°C. Do not freeze/thaw more than 3 times.

Polybrene® reagent not included during
transduction

Transduce the lentiviral construct into cells in the presence of Polybrene® reagent.

Here are possible causes and solutions:

Cause

Solution

Too little antibiotic used for selection

Increase amount of antibiotic.

Selection performed on confluent cells

Before adding selective medium, trypsinize transduced cells and replate in a larger tissue culture plate.

Viral supernatant not diluted sufficiently

Titer lentivirus using a wider range of 10-fold serial dilutions (e.g., 10-2 to 10-8).

Here are possible causes and solutions:

Cause

Solution

Promoter silencing

The CMV promoter is prone to silencing, especially in mouse or rat cells. Screen multiple antibiotic-resistant clones and select the one with the highest expression levels. To avoid the CMV promoter, you may use a promoterless lentiviral vector and choose an alternate promoter such as EF1alpha.

Viral stocks stored incorrectly

Aliquot and store stocks at –80°C. Do not freeze/thaw more than 3 times.

Here are possible causes and solutions:

Cause

Solution

Low transduction efficiency:

  • Polybrene® reagent not included during transduction
  • Non-dividing cell type used
  • Transduce the lentiviral construct into cells in the presence of Polybrene® reagent.
  • Transduce your lentiviral construct into cells using a higher MOI.

MOI too low

Transduce your lentiviral construct into cells using a higher MOI.

Too much antibiotic used for selection

Determine the antibiotic sensitivity of your cell line by performing a kill curve. Use the minimum antibiotic concentration required to kill your untransduced cell line.

Antibiotic used too soon after transduction

Allow at least 48–72 hours after transduction before applying antibiotic selection.

Cells harvested too soon after transduction

Do not harvest cells until at least 48–72 hours after transduction to allow expressed protein to accumulate in transduced cells.

Gene of interest is toxic to cells

Generating constructs containing activated oncogenes or potentially harmful genes is not recommended.

Rearrangement in the LTR regions of the expression construct plasmid DNA

Perform restriction digestion using a combination of Afl II and Xho I. Afl II sites are present in both LTRs. The Xho I site is present in the plasmid backbone at the 3’ end of the insert. Assuming there are no Afl II or Xho I sites in the insert, 3 DNA fragments are generated from the Afl II + Xho I digest. Any unexpected DNA fragments are a result of LTR recombination.

Here are possible causes and solutions:

Cause

Solution

Large volume of viral supernatant used for transduction

  • Remove the “spent” medium containing virus and replace with fresh, complete medium.
  • Concentrate the virus.

Cells are sensitive to Polybrene® reagent

Verify the sensitivity of your cells to Polybrene® reagent. If cells are sensitive, omit the Polybrene® reagent during transduction. DEAE dextran at a concentration of 6–10 μg/mL can be used as an alternative if Polybrene® reagent is toxic to your particular cell type. Optimization of Polybrene® reagent or DEAE dextran for your cell type is critical to success of transduction.

Too much antibiotic used for selection

Determine the antibiotic sensitivity of your cell line by performing a kill curve. Use the minimum antibiotic concentration required to kill your untransduced cell line.

Antibiotic used too soon after transduction

Allow at least 48–72 hours after transduction before applying antibiotic selection.

Gene of interest is toxic to cells

  • Generating constructs containing activated oncogenes or potentially harmful genes is not recommended.
  • Try a different cell line.
  • Transduce at a lower MOI.

Here are possible causes and solutions:

Cause

Solution

Incorrect filter set on cell culture fluorescence microscope or incorrect detection parameters for flow cytometer

Make sure you are using a FITC or Omega XF100 filter set on your inverted fluorescence microscope (see page 7 of the manual) or the FITC detection parameters on your flow cytometer.

Viral stocks stored incorrectly

Aliquot and store stocks in cryovials at –80°C. Do not freeze/thaw more than 3 times.

Polybrene® reagent not included during
transduction

Transduce pLenti6.2-GW/EmGFP into cells in the presence of Polybrene® reagent.

Too soon to see EmGFP expression

For optimal EmGFP expression, wait 4 days post-transduction.

Here are possible causes and solutions:

Cause

Solution

Low transduction efficiency:

  • Polybrene® reagent not included during transduction
  • Non-dividing cell type used
  • Transduce pLenti6.2-GW/EmGFP into cells in the presence of Polybrene® reagent.
  • Transduce pLenti6.2-GW/EmGFP into cells using a higher MOI.

 

MOI too low

Transduce pLenti6.2-GW/EmGFP into cells using a higher MOI.

Cells harvested too soon after transduction

Do not harvest cells until at least 48–72 hours after transduction to allow EmGFP to accumulate in transduced cells.

Here are possible causes and solutions:

Cause

Solution

Promoter silencing

pLenti6.2-GW/EmGFP may integrate into a chromosomal region that silences the CMV promoter controlling expression of EmGFP. Screen multiple antibiotic-resistant clones and select the one with the highest expression levels.

Incorrect filter used with cell culture fluorescence microscope or detection parameters for flow cytometer

Make sure you are using a FITC or Omega XF100 filter set on your inverted fluorescence microscope (see page 7 of the manual) or the FITC detection parameters on your flow cytometer.

This can happen due to excess blasticidin used during selection. Determine the antibiotic sensitivity of your cell line by performing a kill curve. Use the minimum antibiotic concentration required to kill your untransduced cell line.

Here are possible causes and solutions:

Cause

Solution

Incorrect filter set on cell culture
fluorescence microscope or incorrect detection parameters for flow cytometer

Make sure you are using a FITC or Omega XF100 filter set on your inverted fluorescence microscope (see page 7 of the manual) or the FITC detection parameters on your flow cytometer.

Viral stocks stored incorrectly

Aliquot and store stocks in cryovials at –80°C. Do not freeze/thaw more than 3 times.

Polybrene® reagent not included during
transduction

Transduce pLenti6.3/V5-GW/EmGFP into cells in the presence of Polybrene® reagent.

Too soon to see EmGFP expression

For optimal EmGFP expression, wait 4 days post-transduction.

Here are possible causes and solutions:

Cause

Solution

Low transduction efficiency:

  • Polybrene® reagent not included during transduction
  • Non-dividing cell type used
  • Transduce pLenti6.3/V5-GW/EmGFP into cells in the presence of Polybrene® reagent.
  • Transduce pLenti6.3/V5-GW/EmGFP into cells using a higher MOI.

 

MOI too low

Transduce pLenti6.3/V5-GW/EmGFP into cells using a higher MOI.

Cells harvested too soon after transduction

Do not harvest cells until at least 48–72 hours after transduction to allow EmGFP to accumulate in transduced cells.

Here are possible causes and solutions:

Cause

Solution

Promoter silencing

pLenti6.3/V5-GW/EmGFP may integrate into a chromosomal region that silences the CMV promoter controlling expression of EmGFP. Screen multiple antibiotic-resistant clones and select the one with the highest expression levels.

Incorrect filter used with cell culture fluorescence microscope or detection parameters for flow cytometer

Make sure you are using a FITC or Omega XF100 filter set on your inverted fluorescence microscope (see page 7 of the manual) or the FITC detection parameters on your flow cytometer.

This can happen due to excess blasticidin used during selection. Determine the antibiotic sensitivity of your cell line by performing a kill curve. Use the minimum antibiotic concentration required to kill your untransduced cell line.

With the HiPerform™ FastTiter™ lentiviral vectors, the fluorescence signal intensity of EmGFP in the cells is not optimal for visual evaluation using fluorescence microscopy. With these vectors, we recommend using only flow cytometry to detect the EmGFP in transduced cells.

Here are possible causes and solutions:

Cause

Solution

Lenti3.3/TR construct integrated into an inactive region of the genome

Screen other Geneticin®-resistant colonies. Choose the clone that exhibits the highest level of Tet repressor expression for use as the host for your Lenti6.3/TO/V5-DEST construct.

Transduced Lenti3.3/TR into a mammalian cell line in which the CMV promoter is downregulated

Use another mammalian cell line for transduction.

Here are possible causes and solutions:

Cause

Solution

Cells harvested and assayed too soon after addition of tetracycline

  • Culture cells for a longer period of time after addition of tetracycline before assaying for recombinant protein expression. Do not harvest cells until at least 24 hours after addition of tetracycline.
  • Placing cells under Geneticin® selection can improve protein expression results by killing untransduced cells.

Lenti6.3/TO/V5-DEST lentiviral stock not titered

Titer the lentiviral stock using the procedure on page 21 of the manual before use.

Lenti6.3/TO/V5-DEST lentiviral stock stored incorrectly

  • Aliquot and store stocks at –80°C.
  • Do not freeze/thaw more than 3 times.
  • If stored for longer than 6 months, re-titer stock before use.

Here are possible causes and solutions:

Cause

Solution

Did not transduce the Lenti6.3/TO/V5-DEST lentiviral construct into a Tet repressor–expressing cell line

  • Generate a ViraPower™ T-REx™ cell line first, and then use this cell line as the host for the Lenti6.3/TO/V5-DEST virus.
  • Perform the co-transduction procedure (see pages 27–30 of the manual). Make sure that the Lenti3.3/TR lentivirus is transduced into mammalian cells at least 24 hours before transduction of the Lenti6.3/TO/V5-DEST lentivirus.

Forgot to add tetracycline

To induce expression of the gene of interest after transduction of Lenti6.3/TO/V5-DEST lentivirus, add tetracycline to a final concentration of 1 g/mL. Wait for at least 24 hours before assaying for recombinant protein expression.

This can happen if the Lenti6.3/TO/V5-DEST construct is transduced into cells that do not express the Tet repressor. Generate a ViraPower™ T-REx™ cell line first, and then use this cell line as the host for the Lenti6.3/TO/V5-DEST virus. You may also want to confirm that your cells are not being grown in medium with FBS that has high tetracycline levels in it.

Here are possible causes and solutions:

Cause

Solution

Transduced Lenti3.3/TR viral construct at too low of an MOI when compared to the expression construct

Transduce the Lenti3.3/TR viral construct into mammalian cells at a higher MOI (e.g., MOI = 10) than the expression construct (e.g., MOI = 1–5).

Did not wait for a sufficient amount of time after transducing the Lenti3.3/TR viral construct before transducing the Lenti6.3/TO/V5-DEST viral construct

Transduce mammalian cells with the Lenti6/TR construct, and then wait for 24 hours before transducing cells with the Lenti6.3/TO/V5-DEST construct.