Biology Reference
In-Depth Information
10-cm tissue culture dish, and incubate at 37 °C in a CO
2
incubator overnight. Approximately 24 h after plating, cells
can be either transfected with DNA directly or passaged once
at a 1:4 ratio before transfection (
see
Note 5
).
To analyze the immortalizing and transforming functions of human
Ad
E1
and
E4
oncogenes, plasmids expressing the individual (viral)
gene products (e.g., pcDNA3 derivatives encoding E1A-12S,
E1A-13S, E1B-55 kDa, E1B19kDa,
E4orf3
, or
E4orf6
) or combi-
nations thereof (e.g., pAd5
Xho
I-C 15 carrying a viral genomic
fragment comprising the entire Ad5
E1
region) are transfected into
BRK cells. The use of plasmids carrying a selectable marker gene
(e.g., the
neo
marker, which allows for selection with G418) facili-
tates clearing of nontransfected cells and abortive foci induced by
E1A
-mediated transient cell proliferation, thus markedly reducing
background. However, selection for a marker gene eliminates not
only transient but also stably transformed foci derived by “hit-and-
run” transformation mechanisms that do not require permanent
maintenance of oncogenes [
10
]. For cooperative transformation
with strong transforming genes such as combinations of
E1A
with
activated
ras
, the use of a dominant selection procedure is not gen-
erally needed. We routinely transfect BRK cells using a modifi ed
calcium phosphate protocol (
see
Note 6
). Make sure to prepare
duplicate or triplicate samples.
3.2 Transfection
of BRK Cells
1. Mix 1-5
g of plasmid DNA containing the transforming
gene(s) with carrier DNA to give a total DNA amount of
20
μ
μ
g. Add 50
μ
L of 2.5 M CaCl
2
solution and make up to
500
L with sterile, double-distilled water in a 1.5-mL reac-
tion tube. Place 500
μ
L of 2× HeBS buffer into a 5-mL
(12 × 75 mm) polystyrene round-bottom snap cap tube.
2. Transfer the DNA-CaCl
2
mixture drop by drop to the 500
μ
L
of 2× HeBS (with a 1-mL pipet and a plastic tip) while vortex-
ing HeBS at medium speed.
3. Allow precipitate to sit for 20 min at room temperature.
4. Add the total volume (1 mL) of the precipitate to each 10-cm
dish of cells containing 10 mL growth medium and mix
gently.
5. Incubate at 37 °C in a CO
2
incubator for at least 5 h or
overnight.
6. Gently shake the dish to dislodge the residual precipitate and
remove the medium. Wash once with 10 mL of PBS, feed with
10 mL of fresh medium, and incubate at 37 °C.
7. Optional: add G418 (500
μ
g/mL) within the next few days.
8. Change medium every fourth day. In assays involving stronger
transforming genes, such as the activated
ras
oncogene, the
μ
