Biology Reference
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9. For further cultivation use medium supplemented with
selection antibiotics.
10. 2-3 weeks post transduction the cells can be stained with
crystal violet.
3.5 Staining of
Immortalized Colonies
and Transformed Foci
The immortalized/transformed colonies become visible approx
1 week after transfection. After periodical visual examination of the
colonies, if the sizes of the colonies appear satisfactory, stain with
crystal violet (or Giemsa) for quantitation and photography.
1. 1 day prior to staining, feed cells with fresh medium.
2. Remove medium and wash once with 5 mL PBS.
3. Fix and stain the colonies with crystal violet solution for 5 min,
and wash two times with tap water.
4. Dry the dishes, count the colonies, and photograph if
necessary.
5. The immortalized and transformed colonies obtained with
E1A/E1B
and
E1A/E1B
plus
E4orf6
are shown in Figs.
2
and
3
.
3.6 Characterization
of Transformed Cells
To determine the extent of transformation, the transformed cells
are generally assayed for their ability to form anchorage- independent
colonies on semi-solid (soft-agar) medium in vitro and/or exam-
ined for their ability to form tumors in immunodefi cient mice or
immunocompetent syngeneic hosts. Here we describe simple
tumorigenicity and malignancy assays in athymic nude mice.
Fig. 2
Focus formation by (
a
) Ad5
E1A
and
E1B
, or (
b
)
E1A
,
E1B
, and
E4orf6
. Baby
rat kidney cells were transfected with 5
g each of plasmids expressing
E1A/
E1B
and
E4orf6
as indicated using the calcium phosphate precipitation tech-
nique. Transfected cells were stained with crystal violet 27 days after transfec-
tion and scanned
μ
