Biology Reference
In-Depth Information
cationic molecules can also be used. For reasons of effi ciency,
simplicity, and cost, PEI transfection is highly recommended.
In the case that PEI is used, the following protocol for the
preparation of the PEI/DNA complex can be applied:
(a) Prepare PEI and/DNA complexes in 2-mL Eppendorf tubes:
(a.1) In a tube labelled A: put 6
μ
g DNA and 150
μ
L of
sterile 150 mM NaCl. Mix well.
(a.2) In a tube labelled B: put 13.5
μ
L PEI 10 mM and
L of sterile 150 mM NaCl. Mix well.
(b) Slowly add solution B dropwise to solution A.
(c) Incubate the DNA with the transfection reagent for
20 min at room temperature.
(d) Remove growth medium from HEK-293 cells and wash
once with serum-free DMEM gently. Remove DMEM
and add 0.5 mL of serum-free DMEM per well.
(e) Add PEI/DNA complexes dropwise to cells. Incubate at
37 °C and 5 % CO
2
for 6 h.
(f) Remove medium and add 3 mL of fresh DMEM + 10 % FBS.
8. If the recombinant viral genome carries a fl uorescent marker
gene, check initial transfection effi ciency as well as adenoviral
infection during amplifi cation. It is not expected to observe a
visible cytopathic effect (CPE) in this step.
9. The adenovirus life cycle spans 36 h. In our conditions, CPE
begins at around 30 h postinfection, peaking at approximately
40 h, which is when we usually harvest the virus. Some factors,
including the producer cell line and the gene of interest
expressed from the adenoviral genome, may change the CPE
appearance pattern, resulting in variability in harvest times.
10. Inoculate 7.5
150
μ
l of lysate in A549 nonpermissive human cells
and culture in the absence of antibiotics for several days (usu-
ally 7 days), to amplify the possible mycoplasma contamination
and increase sensitivity. Then, Venor™ GeM Mycoplasma
Detection Kit can be used to detect
Mycoplasma
following the
instructions of the manufacturer.
11. Repeated freeze-thaw cycles should be avoided as they will
cause a decrease in viral infectivity. Aliquot in small volumes to
minimize this effect.
12. HEK 293 cells are normally used for infectious unit quantifi ca-
tion. The detection of RCAs requires the use of noncomple-
mentary human cells such as A549, unlike replication-defective
adenoviruses, RCAs will be able to replicate in A549 cells.
13. The range of dilutions will be selected according to the esti-
mated concentration of the viral stock to titer. A standard
scheme of serial dilutions is drawn in Table
1
.
μ
