Supporting Materials and Methods
Plasmids
pT/KRas-G12V was constructed by inserting a KRas-G12V fragment into the
pT3/EF1α plasmid. The corresponding fragment was generated by PCR from the
plasmid
pORF9-mKRas2
(InvivoGen)
using
ATGACTGAGTATAAACTTGTGGTGGTTGGAGCT
the
primers
G12V-fw:
GTTG
and
G12V-rev:
GTATTCACATAACTGTACACCTTG. pT/KRas-G12V-IRES-GFP was cloned by
coupling IRES-GFP (pQCXIX, Clontech) with KRas-G12V into pT/KRas-G12V.
pT/EF1α-EGFP was designed by cloning of EGFP into pT3/EF1α. The plasmid
pT/mAlb-EGFP was constructed by replacing the EF1α-promoter in pT/EF1α-EGFP
by the mouse albumin-promoter from the plasmid pAlb.GH (kindly provided by Mark
A. Magnuson, Vanderbilt University). To construct pT/mCK19-EGFP, the murine
CK19-promoter
was
amplified
by
PCR
using
GTTCCTTTCTAAGACCCACCAGTC
the
primers
and
CK19-fw:
CK19-rev:
GAGACCAGAGCGAAGGAAAAAG. The resulting fragment was inserted into
pT/EF1α-EGFP thus replacing the EF1α-promoter.
Detection of Cre-mediated Trp53Δ2-10-recombination in tumor cells
Induced tumors were isolated, dissected, and incubated 30 min at 37 °C in
RPMI1640+Glutamax
(Life
Technologies)
supplemented
with
200µg/ml
of
collagenase IA, collagenase IV, and hyaluronidase IV, 300µg/ml dispase, and
50µg/ml DNase I (all Sigma). Separated cells were filtered through a 40µm strainer,
and cultured under standard conditions. After 8 passages, cells were counted and
single cells were seeded in 96-well plates by limiting dilution. Growing colonies were
analyzed for RT-qPCR or p53floxgenotyping. DNA was isolated using QIAmp DNA
Mini Kit (Qiagen). Primers for detection of Trp53F2-10 alleles by PCR were described
previously (1). For detection of the 5´loxP-site, intron 1 PCR yields a 370 bp or 288
bp fragments for the floxed or wildtype alleles, respectively and for the 3´loxP site,
intron 10 PCR yields a 584 bp or 431 bp fragments for floxed or wildtype alleles,
respectively. In case of recombination of Trp53Δ2-10 allele, 612-bp fragment can be
observed. For detection of control exon 11, PCR yields a 390 bp fragment.
Reverse Transcription qPCR
Total RNA was isolated using the RNeasy-Mini-Kit (Qiagen). Random hexamer
primer and TaqMan RT-Reagents (Applied Biosystems) were used to generate
cDNA. For quantification, SYBR-Green (Eurogentec) and Lightcycler®480II (Roche)
was used. Ct-values of amplicons were normalized by GAPDH as internal control.
Primers for EGFP (2) and p53 (3) quantification were described previously. Following
primers
were
used
for
mKRas
and
mGAPDH
quantification:
KRas-fw:
GTAAAGGACTCTGAAGATGTGCC; KRas-rev: CAATGAACGGAATCCCGTAACTC;
GAPDH-fw:
AGAACATCATCCCTGCATCC;
GAPDH-rev:
CACATTGGGGGTAGGAACAC.
References
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suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer. Nat
Genet 2001 Dec;29(4):418-425.
2. Lalancette C, Thibault C, Bachand I, Caron N, Bissonnette N. Transcriptome analysis of bull
semen with extreme nonreturn rate: use of suppression-subtractive hybridization to identify
functional markers for fertility. Biol Reprod 2008 Apr;78(4):618-635.
3. Chang CJ, Chao CH, Xia W, Yang JY, Xiong Y, Li CW, et al. p53 regulates epithelial-mesenchymal
transition and stem cell properties through modulating miRNAs. Nat Cell Biol 2011
Mar;13(3):317-323.