Biology Reference
In-Depth Information
formation of metastatic tumor nodules in the internal organs such
as lungs or lymph nodes. If the burden of primary tumors is heavy
for the animals (tumor area
100 mm
2
), the primary tumors must
be surgically removed, after which the animals are maintained for
an additional 2-4 weeks and autopsied.
≥
4
Notes
1. The 2× HeBS solution can be rapidly tested by mixing 0.5 mL
of 2× HeBS with 0.5 mL of 250 mM CaCl
2
and vortexing.
A fi ne precipitate should develop that is readily visible in the
microscope.
2. Neonatal Fisher rats are equally suitable. Kidneys may also be
obtained from animals between 2 and 4 days after birth, which,
however, will result in reduced tissue yields.
3. During preparation of BRK cells, as much of the capsule pres-
ent in the kidney should be removed as possible. If it is not
fully removed, the preparation will contain fi broblasts.
Fibroblasts will grow faster than epithelial cells, causing
increased background.
4. Try to minimize incubation periods because prolonged diges-
tion of minced kidneys with collagenase-dispase will result in
reduced viability and reduced plating effi ciency of epithelial
cells.
5. Continued passaging before transfection will result in decreased
transformation effi ciencies.
6. Other transfection methods including liposome-mediated
techniques may also be employed. BRK cells are also highly
suited for gene transduction with adequate retroviral vectors.
7. The use of lentiviral vectors with a selectable marker gene (e.g.,
the neo and bsr marker, which allows for selection with G418
and blasticidin S) facilitates clearing of non-transduced cells
and abortive foci induced by E1A-mediated transient cell pro-
liferation, thus markedly reducing background.
References
1. Endter C, Dobner T (2004) Cell transforma-
tion by human adenoviruses. Curr Top
Microbiol Immunol 273:163-214
2. Williams JF, Zhang Y, Williams MA, Hou S,
Kushner D, Ricciardi RP (2004) E1A-based
determinants of oncogenicity in human adeno-
virus groups A and C. Curr Top Microbiol
Immunol 273:245-288
3. Chinnadurai G (2004) Modulation of onco-
genic transformation by the human adenovirus
E1A C-terminal region. Curr Top Microbiol
Immunol 273:139-161
4. White E (2001) Regulation of the cell cycle
and apoptosis by the oncogenes of adenovirus.
Oncogene 20:7836-7846
5. Täuber B, Dobner T (2001) Adenovirus early
E4 genes in viral oncogenesis. Oncogene
20:7847-7854
6. Glaunsinger BA, Weiss RS, Lee SS, Javier RT
(2001) Link of the unique oncogenic properties
