Adenoviral Gene Delivery for Mammalian Expression—Troubleshooting

Solution

Incorrect antibiotic used to select for transformants

Select for transformants on LB agar plates containing 100 μg/mL ampicillin.

LR recombination reaction not treated with proteinase K

Treat reaction with proteinase K before transformation.

Too much entry clone DNA used in the LR reaction

Use 50–150 ng of the entry clone in the LR reaction.

Inappropriate ratio of entry clone:DEST vector used in the LR reaction

Aim for a 1:1 molar ratio of entry clone:DEST vector.

LR recombination of >5 kb insert only incubated for 1 hr

For inserts larger than 5 kb, we recommend to incubate the LR reaction overnight. Note: This overnight incubation will also boost colony count for smaller inserts.

Adenoviral Destination vector DNA was sheared

Use care when handling the adenoviral Destination vector. Do not perform excessive manipulations (e.g.,vortexing or pipetting the solution vigorously) that may shear the DNA.

Didn’t use the suggested amount of LR Clonase® II enzyme mix or LR Clonase® II enzyme mix was inactive

-Make sure to store the LR Clonase® II enzyme mix at –20°C.
-Do not freeze/thaw the LR Clonase® II enzyme mix more than 10 times.
-Use the recommended amount of LR Clonase® II enzyme mix (see page 14 of the manual).
-Test another aliquot of the LR Clonase® II enzyme mix.

Not enough LR reaction transformed

Transform 2–3 μL of the LR reaction into the appropriate competent E. coli strain. Use E. coli cells with a transformation efficiency >1 x 108 cfu/μg.

Not enough transformation mixture plated

Increase the amount of E. coli plated.

Solution

LR reaction transformed into an E. coli strain containing the F′ episome and the ccdA gene

Use an E. coli strain that does not contain the
F’ episome for transformation (e.g.,TOP10,
DH5α-T1R).

Deletions (full or partial) of the ccdB gene from adenoviral Destination vector

The adenoviral Destination vectors are provided in solution and are ready to use in an LR reaction. However, if you wish to propagate them, we recommend using One Shot® ccdB Survival™ 2 T1R Chemically Competent Cells (Cat. No. A10460).
-Select for transformants in media containing 50–100 μg/mL ampicillin and 15–30 μg/mL chloramphenicol, to maintain the integrity of the vector.
-Prepare plasmid DNA from one or more colonies and verify the integrity of the vector before use.

Solution

Low transfection efficiency:
-Adenoviral Destination vector DNA was sheared
-Incomplete Pac I digestion or digested DNA contaminated with phenol, ethanol, or salts
-Unhealthy 293A cells; cells exhibit low viability
-293A cells plated too sparsely on the day before transfection
-Plasmid DNA:transfection reagent ratio incorrect

-Use care when handling the adenoviral Destination vector. Do not perform excessive manipulations (e.g.,vortexing or pipetting the solution vigorously) that may shear the DNA.
-Repeat the Pac I digestion. Make sure purified DNA is not contaminated with phenol, ethanol, or salts.
-Use healthy 293A cells; do not overgrow cells.
-Cells should be 90–95% confluent at the time of transfection.
-Optimize such that plasmid DNA (in μg):Lipofectamine® 2000 (in μL) ratio ranges from 1:2 to 1:3. If you are using another transfection reagent, optimize according to the manufacturer’s recommendations.

Viral supernatant too dilute

Concentrate virus using CsCl purification or any method of choice.

Viral supernatant frozen and thawed multiple times

Do notfreeze/thaw viral supernatant more than 10 times.

Gene of interest is large

Viral titers generally decrease as the size of the insert increases; inserts larger than 6 kb (for pAd/CMV/V5-DEST™) and 7.5 kb (for pAd/PL-DEST™) are not recommended.

Gene of interest is toxic to cells

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

Solution

Viral stocks stored incorrectly

Aliquot and store stocks at –80°C. Do not freeze/thaw more than 10 times.

Incorrect titering of cell line used

Use the 293A cell line or any cell line with the characteristics discussed on page 23 of the manual.

Agarose overlay incorrectly prepared

Make sure that the agarose is not too hot before addition to the cells; hot agarose will kill the cells.

Viral stock with very low titer or very high titer

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

Solution

Viral stocks stored incorrectly

Aliquot and store stocks at –80°C. Do not freeze/thaw more than 10 times.

Gene of interest contains a Pac I site

Perform mutagenesis to change or remove the PacI site.

Solution

Poor transduction efficiency:
-Mammalian cells not healthy
-Non-dividing cell type used

-Make sure that your cells are healthy before transduction.
-Transduce your adenoviral construct into cells using a higher MOI.

MOI too low

Transduce your adenoviral construct into cells using a higher MOI.

Low viral titer

Amplify the adenoviral stock using the procedure on page 20 of the manual.

Adenoviral stock contaminated with RCA (replication-competent adenovirus)

-Screen for RCA contamination.
-Prepare a new adenoviral stock or plaque purify to isolate recombinant adenovirus.

Cells harvested too soon after transduction

Do not harvest cells until at least 24 hours after transduction.

Cells harvested too long after transduction

For actively dividing cells, assay for maximal levels of recombinant protein expression within 5 days of transduction.

Gene of interest is toxic to cells

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

Solution

Too much crude viral stock used

-Reduce the amount of crude viral stock used for transduction ordilute the crude viral stock.
-Amplify the adenoviral stock.
-Concentrate the crude viral stock.

Wild-type RCA (replication-competent adenovirus) contamination

Screen for RCA contamination. Plaque purify to isolate recombinant adenovirus or prepare a new adenoviral stock.

Gene of interest is toxic to cells

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