Biology Reference
In-Depth Information
thus can save time. 116 cells should be subcultivated when they
reach 90 % confl uency and it is strongly recommended to never let
them become superconfl uent. 116 cells are cultivated in 150 mm
dishes in supplemented MEM. Use 30 mL of supplemented
medium per 150 mm dish. Subcultivation is required at a ratio of
1:3 every 3 days. Pre-warmed media, trypsin, and PBS (37 °C) are
recommended for subcultivation.
1. Remove medium by aspiration.
2. Wash cells with 10 mL pre-warmed PBS. Take care to carefully
pipette the PBS in order to not detach the cells from the dish.
3. Aspirate PBS.
4. Add 3 mL trypsin to cells. Trypsinize for 1 min at room
temperature.
5. Carefully aspirate trypsin.
6. Resuspend the cells in fresh medium and transfer the appropri-
ate volume to new dishes.
Subcultivated 116 cells should be allowed to attach to the new
dishes for 48 h prior to infection/transfection for rescue/amplifi -
cation purposes.
To rescue HC-Ad vectors from their corresponding vector plasmid,
the plasmid is linearized, transfected into 116 cells and the cells are
coinfected with helper virus. The initial transfection effi ciency
determines to a signifi cant degree the number of amplifi cations that
are required to obtain a high-titer HC-Ad vector preparation.
Optimizing transduction effi ciencies is thus mandatory (
see
Note 1
).
3.2 HC-Ad
Vector Rescue
1. For each vector to be rescued seed 116 cells into one 60-mm dish
in a way that they reach a confl uency of 70-80 % within 48 h.
2. Linearize 15-20
g of the HC-Ad vector plasmid with the
appropriate enzyme in a total volume of 100
μ
L.
3. Purify the linearized plasmid by phenol/chloroform extraction
and ethanol precipitation (
see
Note 2
).
4. Perform agarose gel electrophoresis and ethidium bromide
staining with 100 ng of the linearized and purifi ed plasmid.
Only two bands should be visible: a large one comprising the
HC-Ad vector genome and a smaller one comprising the bac-
terial backbone (
see
Note 3
).
5. 30 min prior to transfection replace the medium of the cells to
be transfected with 2 mL of fresh, supplemented MEM.
6. Transfect the cells (one 60-mm dish per vector) with 4
μ
g of
the linearized and purifi ed HC-Ad vector plasmid using jetPEI
according to the manufacturer's instructions.
7. One hour after transfection infect the cells with 15-20 MOI of
helper virus (
see
Notes 4
and
5
).
μ
