Biology Reference
In-Depth Information
primary fi broblasts and immortalized cell lines. First, the use of
BRK cells results in negligible background transformation. Second,
because human neoplasia are predominantly (approx. 90 %) epi-
thelial in origin, the results obtained with BRK (epithelial) cells
may be extrapolated to human cancers.
In 2000, Schiedner and coworkers published, that primary
human amniotic fl uid cells (AFC) are susceptible to Ad5
E1-mediated transformation [
11
]. Although AFCs are an unspe-
cifi c mixture of primary human cell types, they can nevertheless be
easily obtained after amniocentesis and cultured for at least a
couple of weeks in vitro. Therefore, they are quite suitable to study
Ad-mediated transformation in a human cell system.
2
Materials
1. Baby rats: a litter (approx 6-12 pups) of 5-7-day-old neonatal
outbred Sprague Dawley (SD) rats can be purchased from
Harlan SD (Indianapolis, IN).
2. Primary human amniotic fl uid cells (AFC) can be easily iso-
lated from the amniotic fl uid after amniocentesis for clinical
purposes and further cultivated in vitro [
11
].
3. Nude mice: athymic NMRI (nu/nu) mice can be obtained
from Harlan SD.
4. Plasmids: mammalian expression plasmids (e.g., pcDNA3
derivatives) encoding Ad E1A12S/13S, E1B-55 kDa, E1B-
19 kDa, E4orf3, E4orf6, and activated
ras
have been described
[
8
,
9
] and can be obtained from the authors.
5. Carrier DNA: purifi ed DNA from fi sh sperm can be obtained
from Roche. Resuspend DNA in sterile double-distilled water at
10 mg/mL and shear to an average fragment length of 10,000 bp
(evaluated by agarose gel/ethidium bromide electrophoresis) by
sonication. Store in aliquots at −20 °C. Dilute 1:10 to make
working stocks (1 mg/mL).
6. Lentiviral gene ontology vectors (e.g., LeGO-iV2/Cer2 deri-
vates; Addgene, cat. no. 27334, 27338) encoding Ad
E1A12S/13S, E1B-55 kDa, E1B-19 kDa, E4orf3, and E4orf6
can be obtained from the authors.
7. Packaging plasmids pMDLg/pRRE, pRSV-Rev, and pMD2.G
can be purchased from Addgene (Addgene; cat. no. 12251,
12253,12259).
8. Producer cell line HEK293T (ATCC; cat.no. CRL-11268TM).
9. Phosphate-buffered saline (PBS) without Ca
2+
and Mg
2+
: dis-
solve 8.0 g NaCl, 0.20 g KCl, 1.60 g Na
2
HPO
4
(or 1.78 g
Na
2
HPO
4
2H
2
O), and 0.24 g KH
2
PO
4
in 900 mL of double-
distilled water and adjust the pH to 7.2-7.4 with HCl or
⋅
