Hormone-Induced Protection of Mammary Tumorigenesisin Genetically Engineered Mouse Models - Genetic Engineering: Recent Developments in Applications - page 272

Sigma) was packed into the Silastic capsules also in a cellulose matrix. All these
capsules were primed by soaking overnight in medium 199 (Gibco, Grand Island,
NY, USA) at 37°C. Silastic capsules were implanted subcutaneously dorsally.
rnA Analysis
A group (n = 3) of control animals and animals treated with estradiol or
estradiol plus progesterone were killed immediately after the 3 weeks of hor-
mone treatments. Mammary glands were removed from all the groups for the
transgene expression analysis. Another set of animals (n = 15) received the
above-mentioned hormone treatments and were killed at 9 months of age.
Mammary tumor and mammary tissue adjacent to the mammary tumors were
excised, snap-frozen in liquid nitrogen, and stored at -80°C for molecular
analysis. Total RNA isolated from the normal mammary gland, mammary
tumor, and mammary tissue adjacent to the mammary tumor was subjected
to real-time RT-PCR analyses to study the expression of the transgene [20].
RNA was isolated with Trizol reagent, and real-time reverse transcription was
performed with the one-step QuantiTect SYBR Green kit (Qiagen Inc., Va-
lencia, CA, USA) in accordance with the manufacturer's specifications. 18S
RNA was used as standard and positive control in these assays. Fold changes
between samples for relative Her-2/neu transgene mRNA expression were cal-
culated from the differences in ∆ Ct values between the two samples ( ∆∆ Ct)
and the equation Fold change = 2- ∆∆ Ct. Real-time RT-PCR was performed
with the oligonucleotide primers 5'-GGAACCTTACTTCTGTGGTGT-3'
and 5'-GGAAAGTCCTTGGGGTCTTCT-3' targeting the SV40 poly(A) re-
gion of the transgene. Cyclin D1 expression was performed on the mammary
tumors from the control and hormone-treated groups with the oligonucle-
otide primers 5'-CATCAAGTGTGACCCGGACTG-3' and 5'-CCTCCTC-
CTCAGTGGCCTTG-3'.
results
effect of short-term Hormone stimulation on P53-null
Mammary tumorigenesis
The tumorigenic response of the p53-null mammary epithelium exposed to estro-
gen and progesterone combination at 2 to 4 weeks after transplantation is shown
in Figure 1 and Table 1. In the control transplants, 16/66 (24%) produced tumors
by 45 weeks after transplantation, with an initial tumor latency of 20 weeks.
This incidence is consistent with previous studies [10,18]. In the hormone-treated