Generation of a pancreatic cancer model using a Pdx1-Flp recombinase knock-in allele
Generation of a pancreatic cancer model using a Pdx1-Flp recombinase knock-in allele
Jinghai Wu 0 1 2
Xin Liu 0 1 2
Sunayana G. Nayak 0 1 2
Jason R. Pitarresi 0 1 2
Maria C. Cuitiño 0 1 2
Lianbo Yu 2
Blake E. Hildreth 2
Katie A. Thies 2
Daniel J. Schilling 0 1 2
Soledad A. Fernandez 2
Gustavo Leone 2
Michael C. Ostrowski 2
0 Cancer Biology and Genetics Department, The Ohio State University , Columbus , Ohio, United States of America, 3 Center for Biostatistics, Department of Biomedical Informatics, The Ohio State University , Columbus , Ohio, United States of America, 4 Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina, United States of America, 5 Department of Biochemistry and Molecular Biology, Medical University of South Carolina , Charleston, South Carolina , United States of America
1 Comprehensive Cancer Center, The Ohio State University , Columbus, Ohio , United States of America
2 Editor: Francisco X. Real, Centro Nacional de Investigaciones Oncologicas , SPAIN
The contribution of the tumor microenvironment to the development of pancreatic adenocarcinoma (PDAC) is unclear. The LSL-KrasG12D/+;LSL-p53R172H/+;Pdx-1-Cre (KPC) tumor model, which is widely utilized to faithfully recapitulate human pancreatic cancer, depends on Cre-mediated recombination in the epithelial lineage to drive tumorigenesis. Therefore, specific Cre-loxP recombination in stromal cells cannot be applied in this model, limiting the in vivo investigation of stromal genetics in tumor initiation and progression. To address this issue, we generated a new Pdx1FlpO knock-in mouse line, which represents the first mouse model to physiologically express FlpO recombinase in pancreatic epithelial cells. This mouse specifically recombines Frt loci in pancreatic epithelial cells, including acinar, ductal, and islet cells. When combined with the Frt-STOP-Frt KrasG12D and p53Frt mouse lines, simultaneous Pdx1FlpO activation of mutant Kras and deletion of p53 results in the spectrum of pathologic changes seen in PDAC, including PanIN lesions and ductal carcinoma. Combination of this KPF mouse model with any stroma-specific Cre can be used to conditionally modify target genes of interest. This will provide an excellent in vivo tool to study the roles of genes in different cell types and multiple cell compartments within the pancreatic tumor microenvironment.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This work was supported by NIH
(PO1CA097189, MCO and GL); https://www.nih.
gov/. The funders had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
Conditional gene knockout is a powerful tool to study the role of individual genes in living
organisms. This technique eliminates many of the side effects associated with conventional
gene knockout, such as embryonic lethality and the lack of tissue specificity, among others. A
conditional knockout approach controls excision of the endogenous target DNA through
sitespecific recombination (SSR), which involves a recombinase and its short DNA recognition
site. The most commonly used site-specific recombinases include Cre (of bacteriophage P1),
Flp (of the yeast Saccharomyces cerevisiae) and Dre (of bacteriophage D6). These recombinase
enzymes recognize 34 bp Loxp, 34 bp Frt, and 32 bp Rox target sites, respectively, and catalyze
a reciprocal conservative recombination between two identical target sequences. This
recombination results in a sequence deletion between the two target sites. Although initial studies
showed that Cre and Dre were more efficient recombinases than Flp in mammalian cells, a
codon optimized version of Flp (termed FlpO), has greatly improved its reliability [
Because Cre, Flp, or Dre recombinases are not expressed in mammalian cells, there is no risk
of accidental target site recombination in conditional gene knockout mice. Therefore,
conditional gene knockout mice are often used as models of human diseases. It has also
tremendously increased our ability to study complex human diseases in specific developmental stages
and tissues through spatial or temporal gene inactivation via DNA excision. In addition,
through combination of different SSR systems within the same model, we can achieve
spatiotemporal control of distinct genetic ablation/induction because of independent regulation of
PDAC is among the most deadly malignant solid tumors, with over 95% of patients
succumbing to the disease within five years of diagnosis, which is largely the result of no
effective therapies beyond surgery [
]. The most prominent histopathologic hallmark of
pancreatic cancer is its uniquely dense stromal reaction, which consists of activated
fibroblasts, increased amounts of extra-cellular matrix (ECM), immune cell infiltrates, and
abnormal angiogenesis . The stroma undergoes a dramatic expansion in concert with the
stepwise development of PDAC, suggesting that the stroma is an active player in PDAC
progression. This has sparked interest in selectively targeting the tumor stroma to increase
therapeutic efficacy. However, current knowledge of how the stroma influences tumor initiation,
growth, and metastasis remains rudimentary. While current mouse models such as the
LSL-KrasG12D/+;LSL-p53R172H/+;Pdx-1-Cre (KPC) model accurately reflect the genetics of
pancreatic tumor cells and human tumor progression [6±8], these models depend on
epithelial-specific Cre-mediated recombination to drive tumorigenesis. Therefore, concurrent
Cre-loxP recombination in stromal cells is not practical in these models, limiting the in vivo
study of stromal genetics in tumor initiation and progression. Thus, the development of
mouse models that incorporate stroma-specific genetic modifications in an autochthonous
tumor system is crucial to the advancement of the PDAC field. To address this issue, the
ideal strategy would be to combine an alternative recombinase system in epithelial cells with
Cre-loxP in stromal cells, since Frt-STOP-Frt(FSF) KrasG12D and p53frt/frt animals are
available. Therefore, we set to develop a knock-in mouse model termed as Pdx1FlpO, in which the
FlpO gene is specifically inserted into the transcriptional start site of pancreas-duodenum
homeobox 1 (Pdx1). We then combined this Pdx1FlpO mice with the above mentioned lines
to generate a Flp/Frt PDAC mouse model, Pdx1FlpOki;FSF-KrasG12D/+,p53frt/frt (KPF), which
is comparable to the historical KPC tumor model.
A transgenic PdxFlpO mouse model was recently reported [
]. However, two lines of
these PdxFlpO mice showed the transgene located in chromosome 1 and 12, while the normal
physiologic location of the Pdx1 gene is chromosome 5. With these different chromosomal
integration sites, FlpO expression may be influenced by transcriptional regulators other than
authentic pancreas-specific regulators. Therefore, the effects of FlpO recombination on mutant
Kras and p53 activation may have off-target effects other than normal Pdx1 residential tissues
(pancreas, duodenum, and bile duct).
The results presented herein demonstrate the exclusive expression of FlpO in Pdx1
expressing tissues in our knock-in PdxFlpO mouse model. FlpO is only expressed in duodenal
epithelium, pancreatic acinar cells, ductal cells, and islet cells with no expression in the stroma. This
provides an excellent model to investigate genes of interest specifically in stromal cells when
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combined with stroma-specific Cre-loxP. The knock-in p53 heterozygous and homozygous
mice (KPF) develop the spectrum of pathologic changes seen in human PDAC, including
acinar to ductal metaplasia (ADM), pancreatic intraepithelial neoplasia (PanIN) and invasive
ductal carcinoma, with average survival times of 4.5 and 2 months, respectively. Moreover, the
variability of the average survival times is small when compared to published transgenic KPF
models, indicating a highly reliable and reproducible KPF model. In summary, this newly
developed knock-in KPF mouse model is a valuable tool for studying stromal tissue-specific
function of genes of interest in the pancreatic tumor microenvironment.
Generation of Pdx1FlpOki mouse line
Codon usage-optimized Flp (FlpO) cDNA linked with beta-globin polyA was fused into the
start codon site of the Pdx1 gene, replacing the CFP in the previously generated Pdx1CFP
targeting vector [
]. A LoxP-NeoR-LoxP (LNL) fragment was introduced to select neomycin
resistant clones, while a DNA fragment containing mouse phosphoglycerol kinase promoter
(Pgk)-driven expression of thymidine kinase was inserted outside of the short arm for negative
selection. Therefore, this targeting allele contained the mouse Pdx1 long homology arms of
8.1kb, FlpO cDNA/beta-globin polyA, flanked neomycin resistant cassette (LNL), Pdx1 short
arm of 3.6kb and a Pgk-TK cassette, respectively (Fig 1A). Homologous recombination
resulted in replacement of a 4.0 kb region of the Pdx1 locus containing exon 1 with codon
usage-optimized FlpO cDNA, human beta-globin polyA, and a Pgk-driven neomycin
resistance gene flanked by LoxP sites. Clones of interest that had undergone the expected
homologous recombination were identified by a 19.0 kb band after digestion with HindIII after
hybridization with a 5' probe, and a band of 7.2 kb after digestion with AflII hybridized with a
3' probe (ES cell clone #3, Fig 1B). After microinjection of positive ES cell clone WO3 into
blastocysts of C57BL/6 mice, the first generation (F0) of Pdx1FlpOLNL mice was examined by
genotyping PCR using primers targeting allele recombination in both the 5' and 3' arms.
Correctly recombined clones were identified by 0.4 and 1.1 kb bands on the 5' and 3', respectively
(Fig 1C). The LoxP-flanked NeoR sequence in Pdx1FlpOLNL (F0) mice was further removed by
breeding to Sox2-Cre mice. The resultant Pdx1FlpOknockin (denoted PdxFlpOki) allele was
maintained in a mixed background.
In vivo expression specificity test of the Pdx1FlpO allele
To evaluate the expression of FlpO recombinase in multiple tissues, we crossed our Pdx1FlpOki
mouse with the p53frt/frt mouse, of which exons 2±6 of the p53 gene are flanked by two Frt sites
]. After collecting all tissues from Pdx1FlpOki;p53Frt/+ mice, we observed recombination of
the p53 Frt allele in the pancreas, duodenum, and bile duct by PCR (Fig 2A). Spleen, heart,
lung, skin, liver, stomach, colon, and kidney demonstrated no recombination (Fig 2A).
To further validate this model, we crossed Pdx1FlpOki mice with Frt-stop-Frt (FSF)-GFP
reporter mice. Immunohistochemical (IHC) staining for GFP on tissues isolated from
Pdx1FlpOki;FSF-GFP mice revealed GFP expression in the pancreas, duodenum and bile duct
but not in the stomach and other tissues (Fig 2B and data not shown). More specifically, GFP
expression in the adult pancreas is mosaic and present in a fraction of ductal cells, islet cells,
and acinar cells (Fig 2B and Figure A in S1 Fig). This is consistent with the previous reports
using Pdx1-Cre and Pdx1-FlpO transgenic mice. In addition, there was no GFP expression in
control FSF-GFP mice. Taken together, these data suggest that Pdx1FlpO knockin allele
expression is restricted to the pancreas, duodenum, and bile duct.
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Fig 1. Generation of Pdx1FlpOki mice. (A) Schematic representation of the targeting vector, the wild-type Pdx1 locus, the Pdx1-FlpO/
beta globin polyA/LoxP-NeoR-LoxP targeting allele, and the Pdx1FlpOki allele after removal of LNL cassette. Mice containing the
Pdx1FlpOPgk-NeoR allele were bred with Sox2-Cre-expressing transgenic mice to remove the LoxP-flanked NeoR cassette. Restriction
sites: HindIII and AflII. Primer locations: p1, p2, p3, and p4. A Pgk-TX cassette was placed following exon 2 as a negative selectable
marker. (B) Southen blot analysis: Genomic DNA from the aforementioned ES cells was digested by either HindIII or AflII, and hybridized
with DNA probes that bind to either 5' or 3' of Pdx1 locus. (C) PCR analysis on both the 5' (p1 and p3) and 3' (p2 and p4) ends of the
targeting vector, WT FVB/N mouse tail DNA, ES cell clone #3, ES cell clone #1, and tail DNA from the F0 chimera mouse generated from
ES cell clone #3.
Pdx1FlpOki;FSF-KrasG12D/+;p53frt/+ mice develop locally advanced
PDAC and have a significant reduction in survival time
Next, we tried to activate the KrasG12D allele and silence the p53 allele in murine pancreatic
progenitor cells by crossing Pdx1FlpOki, FSF-KrasG12D and p53frt animals. To confirm the
induction of Pdx1 in early pancreatic lesions, we evaluated Pdx1 expression in 3-month old
Pdx1FlpOki;FSF-KrasG12D/+;p53frt/+;FSFG–FP mice. IHC staining for Pdx1 in pancreatic tissue
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Fig 2. In vivo Pdx1FlpOki allele expression specificity. (A) PCR analysis of Pdx1FlpOki mediated
recombination of the p53Frt allele in the indicated tissues of Pdx1FlpOki;p53Frt/+ mice. (B) Representative GFP
IHC staining demonstrates mosaic GFP expression in the pancreas and duodenum, but not the stomach, of
Pdx1FlpOki;FSF-GFP mice. Scale bars = 25 μm.
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Fig 3. Kaplan-Meier survival with p53 inactivation. (A) Genetic strategy used to generate p53 heterozygous and homozygous deletion in
Pdx1FlpOki; FSF-KrasG12D mice. (B) Kaplan-Meier survival curves of the indicated genotypes. Median survival of Pdx1FlpOki;FSF-KrasG12D/+;p53frt/+
or Pdx1FlpOki;FSF-KrasG12D/+;p53frt/frt mice is significantly lower than wild-type (WT) mice (p < 0.001, log-rank test for each pairwise combination).
revealed robust Pdx1 expression in PanIN lesions (Figure B in S1 Fig), indicating that Pdx1
induction occurs in PanIN lesions in our KPF mouse model. Further, losing one p53 allele, i.e.
Pdx1FlpOki;FSF-KrasG12D/+;p53frt/+, results in a marked decrease in median survival time (~4.5
months) when compared with their control littermates (Pdx1FlpOki;FSF-KrasG12D/+ animals)
(Fig 3A and 3B). This entire cohort of mice (n = 20) showed invasive PDAC at necropsy. Loss
of two p53 alleles, i.e. Pdx1FlpOki;FSF-KrasG12D/+;p53frt/frt, resulted in an even greater decrease
in median survival (~2 months). Interestingly, our KPF mouse model showed a significant
reduction in survival time when compared to established transgenic KPF models [
], with a
median survival of 135 days versus 183 days in the KPF model in P53frt/+ animals and a median
survival of 60 days versus 85 days in the KPF model in P53frt/frt animals. This likely results
from our Pdx1FlpO model possessing a knock-in allele instead of transgenic allele that is
present in the KPF model. Histologically, Pdx1FlpOki;FSF-KrasG12D/+;p53frt/+ mice developed
classical PanIN lesions and PDAC. At 2±3 months of age, both low and high grade PanIN lesions
were observed, but by 6 months, invasive carcinoma was present (Fig 4A and 4B). The average
weight of the pancreas in Pdx1FlpOki;FSF-KrasG12D/+;p53frt/+ and Pdx1FlpOki;FSF-KrasG12D/+;
p53frt/frt mice increased significantly compared to Pdx1FlpOki;FSF-KrasG12D/+ mice (See S1
Table for details). However, unlike previously described transgenic KPC and KPF mouse
models, which exhibit metastasis to the liver and lung, we observed negligible metastasis to distant
organs, with no lung and only two liver metastases upon evaluation of 53 of our Pdx1FlpOki;
FSF-KrasG12D/+;p53frt/+ and Pdx1FlpOki;FSF-KrasG12D/+;p53frt/frt mice combined (Table 1).
In the last decade, significant advances have been made in understanding the molecular
mechanisms underlying human pancreatic cancer using genetically-engineered PDAC mouse
models. Compared to xenograft mouse models, these GEM models strongly resemble the
histopathology of human PDAC development, providing a great tool to evaluate preclinical
therapeutic strategies for the treatment of pancreatic cancer.
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Fig 4. p53 knockout accelerates PDAC formation. (A) Representative macroscopic view of pancreata from Pdx1FlpOki;FSF-KrasG12D;p53Frt/+ mice
at 3 and 6 months of age. (B) Representative microscopic H&E stained pancreatic sections from Pdx1FlpOki;FSF-KrasG12D;p53Frt/+ mice at 3 and 6
months. Scale bars = 25 μm.
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All PDAC GEM models are based on Kras activating mutations combined with additional
deletions or mutations of other PDAC suppressors such as p53, Smad4, Tgf-β or PTEN [
]. The most widely used and accepted model is the Pdx1-Cre;LSL-KrasG12D/+; LSL-p53R172H/+
transgenic mouse of which mutated Kras and p53 are specifically expressed in pancreatic
progenitor cells and are driven by the transcription factor Pdx1. However, due to the nature of
Cre-LoxP technology, this model is limited in regards to elucidating the function of other gene
targets in different cell types present within the tumor, e.g. in the stromal mesenchymal and
immune cell subpopulations. More recently, the generation of KPF mice has provided a
solution for this issue. This model uses an alternative Flp/Frt-based recombinase system in the
stroma to drive spontaneous PDAC and possesses similar pathology as the KPC mouse model
. By combining these two complementary recombinase strategies, gene expression can be
manipulated simultaneously in different cell types present within the tumor. This will
contribute significantly to the understanding of the relationship between stroma and PDAC
progression in a more mechanistic manner.
However, both KPC and existing KPF mouse models are transgenic mice. Compared to a
knock-in mouse model which is ªspecifically targetedº, transgenic models are well known for
their ªrandom integrationº±the desired gene could end up anywhere in the host genome and
with an unpredictable amount of copy numbers inserted. This ªrandomnessº of transgenic
models is a limitation due to their unpredictability since the desired gene might be placed
under the influence of another strong promoter with a high copy number. Unpredictable levels
of overexpression could lead to high variability in the magnitude of the disease [
]. Thus, it
might not serve as a reliable and reproducible model of human disease. In addition, studies
have demonstrated the leakiness of the previously reported transgenic PdxCre allele in other
tissues, most notably in gastric and oral mucosa, which has led to the development of
extrapancreatic tumors such as papillomas and adenomas [
In this study, we generated a Pdx1FlpO knock-in mouse line. The Pdx1 gene is expressed in
pancreatic progenitor cells starting at embryonic day (E) 8.5. As development proceeds, Pdx1
becomes highly expressed in β cells with lower levels expressed in acinar and other endocrine
]. The mosaic Cre-mediated recombination that occurs within the pancreas using the
Pdx1 enhancer/promoter is well established [
]. More importantly, expression of Pdx1-Cre
has also been shown in skin keratinocytes [
]. By breeding with the FSF-GFP mouse line, we
verified that our knock-in PdxFlpO demonstrated a similar mosaic expression pattern within
acinar, ductal and islet cells in a lineage-specific manner which is consistent between
littermates. More importantly, in contrast to Pdx1-Cre, we did not observe Pdx1-FlpO expression
in the skin. This finding suggests that the leakiness identified in Pdx1-Cre mice might be due
to its transgenic targeting strategy.
Compared to the existing transgenic KPF model [
], our knock-in KPF mouse had a
decreased survival time. The discrepancies between these two studies could be the timing of
FlpO recombinase expression, extension of recombinase expression, genetic background, etc.
The timing of FlpO expression in the existing transgenic model was not described [
work in the literature demonstrates that the regulatory elements used to generate both PdxCre
and PdxFlpO transgenic alleles were sufficient to drive pancreatic expression of Pdx1 alleles
with the same developmental timing as the endogenous gene [
], although the location of
transgene insertion may affect the timing of expression. Thus, it is difficult to predict when
expression began since this transgene was located in Chromosome 12C1-3 instead of the
physiological locus of Chromosome 5. In contrast, our knock-in FlpO was located in the normal
physiological location of the Pdx1 gene (Chromosome 5). Publications from different labs
characterized the expression pattern of Pdx1 in vivo using knock-in mouse models [
Micallef et al presented a knock-in GFP reporter in the endogenous Pdx1 locus, a strategy
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similar to our Pdx1FlpO model [
]. Their pattern of GFP expression was identical to that
reported for the endogenous Pdx1 gene (E9.5, in the dorsal and ventral pancreatic anlage)
[20±22], with GFP not being detected in other embryonic tissues. In addition, similar
phenotypes were observed in a well-characterized Pdx1CFP mouse line [
], in which CFP was also
inserted into exon 1 of the Pdx1 locus by homologous recombination. Both Pdx1 and CFP
were detected in the dorsal and ventral endodermal evaginations of the posterior foregut at
E9.5. From E10.5 to E11.5, Pdx1 and CFP were detected throughout the pancreatic epithelium.
They concluded that this faithfully recapitulated the normal physiologic expression of
endogenous Pdx1. Using their knock-in targeting vector Pdx1CFP as a backbone, we successfully
replaced the Pdx1 reporter-cyan fluorescent protein (CFP) with FlpO recombinase in our
knock-in Pdx1FlpO mouse line. Because the literature already extensively documents the
timing of expression of reporter genes knocked into this locus with identical strategies, it is
reasonable to predict that the timing of the initiation of FlpO recombinase expression in our
mouse model should be the same as it is normally for Pdx1 expression in vivo. In addition, in
our adult knock-in Pdx1FlpO mice, Pdx1-induced FlpO recombination resulted in GFP
expression in PanIN lesions. This indicated that Pdx1 induction is present exclusively in the
epithelial lesions of our adult KPF tumor model. These findings are consistent with the
previous reports using Pdx1Cre (7) and Pdx1FlpO transgenic mice [
PDAC is a cancer type in which mutant p53 impacts disease progression. In addition to the
most frequent Kras gene mutation, the p53 gene is also often mutated in human pancreatic
cancer in 50±75% of cases [
], predominantly through missense mutations such as R175H
and R273H. Consistent with a role for mutant p53 in other mouse models, mice bearing
PDAC driven by oncogenic Kras and a mutant p53 allele have shown a greater number of
metastases compared to similar mice bearing a p53 null allele , likely resulting from
upregulation of PDGFR-β [
]. A full spectrum of PDAC tumorigenesis, including PanINs and
carcinoma, were observed in every single mouse in our study cohorts. However, we have
detected minimal metastasis to distant organs (2 liver metastases out of 53 mice), with loss of
either one or both p53 alleles. This is consistent with other reports on cancer metastasis in
mice harboring a p53 null allele, and likely results from ablating the p53 gene instead of
activating p53 mutations in the pancreatic epithelium. Of note, there is no FSF-p53 mutant model
available currently. In the future, generation of the FlpO-activated p53 mutant mouse line
would greatly benefit this alternative KPF pancreatic cancer model to evaluate metastasis.
Taken together, our knock-in Pdx1FlpOki line is more stable and tissue-specific when
compared to existing transgenic mouse lines. Thus, it has the potential to contribute to 1) studies
evaluating therapeutics at different stages of tumor progression; 2) determining the interaction
between multiple tumor suppressors and oncogenes; and 3) the development of new
diagnostic approaches. Notably, this Pdx1FlpO model is not inducible; therefore, it will be desirable to
develop a conditional knock-in model. This latter system would allow investigators to turn on/
off genes using this recombinase at discrete time points, such as in adulthood. Future studies
should also investigate its compatibility with Cre-loxP targeting of genes of interest in stromal
Materials and methods
Pdx1FlpO knock-in targeting
To avoid potential self-recombination by FlpO (Codon usage-optimized Flp), the
FrthygroR-Frt fragment was initially removed from the Pdx1CFP+HygroR exchange vector [
NotI digestion and subsequent re-ligation. A PCR-amplified FlpO fragment from
] was cloned into the ATG start codon site of the Pdx1 gene in the Pdx1CFP vector,
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followed by insertion of a blunted LoxP-Neo-LoxP (LNL) fragment into the MluI site. A
thymidine kinase (TK) cassette was acquired from EcoRI/PvuII digestion of the pLMJ322 vector.
The TK fragment was blunted and ligated into the MluI digested Pdx1FlpO-LNL vector,
which resulted in the final targeting vector. This targeting vector was named
Pdx1FlpOLNL-TK. The sequence of the full construct (denoted JW9) was confirmed (3730 DNA
Analyzer, Applied Biosystems).
Gene targeting was performed following standard protocols by the Genetically Engineered
Mouse Modeling facility at The Ohio State University James Comprehensive Cancer Center.
In brief, 1 mg of targeting vector linearized with NotI was electroporated into mouse S1B6
(hybrid C57Bl/6-129Sv) ES cells. Following neomycin selection, homologous recombination
was verified by Southern blot. A DNA fragment of 19.0 kb that includes most of the long arm,
the FlpO cDNA, the Pgk-neo cassette, and the entire short arm was detected using a 5' probe,
while a HindIII-digested fragment of 15 kb from the WT Pdx1 allele was detected in all ES
cells. In contrast, an AflII-digested fragment of 7.2 kb from the FlpO knockin allele was
detected using a 3' probe and generated a fragment of 4.8 kb from the WT Pdx1 allele.
Germline transmission was achieved following the microinjection of clone WO3 mES cells into
C57BL/6 blastocysts. Chimeras were crossed to Black Swiss.
Other mouse strains and tumor models
KrasFSF-G12D/+ mice were kindly provided by Tyler Jacks. p53frt/+ and RosaLSL-FLF-GFP mice have
been described previously [
]. Unless otherwise stated, animals were on a mixed C57Bl/
6;129Sv;Black Swiss background. PdxFlpOki, FSF-KrasG12D/+ and p53frt/+ were interbred to
obtain PDAC mice (KPF) with activation of oncogenic KrasG12D and deletion of p53 in the
pancreatic epithelium. All animal experimental protocols were approved by the Ohio State
University Institutional Animal Care and Use Committee. Mice were monitored and weighed
weekly. All control mice (Pdx1FlpOki, FSF-KrasG12D/+) and experimental mice (Pdx1FlpOki,
FSF-KrasG12D/+,p53frt/+ and Pdx1FlpOki,FSF-KrasG12D/+,p53frt/frt) were euthanized with carbon
dioxide (CO2) and cervical dislocation by 6 months of age. Mice were removed sooner when
they met Early Removal Criteria (ERC) which included an unkempt hair coat, decreased
alertness and mobility, reduced food and water intake, and a 20% loss of body weight if any of
these symptoms persisted for more than 24 hrs. The overall health of the animals was
monitored by our trained laboratory personnel and the veterinary staff.
Histologic analysis and immunohistochemistry
Dissected mouse pancreata were fixed in 10% neutral-buffered formalin for 48 hr and then
transferred to 70% ethanol. Tissues were processed, embedded in paraffin, cut in 5μm sections
on positively charged slides, de-paraffinized, rehydrated, and stained with H&E. For GFP IHC,
slides were heated at 65ÊC for 15 min and after standard de-paraffinization and rehydration,
sections were unmasked in 1X Target Retrieval solution (pH = 6.0). Endogenous peroxidases
were quenched in 3% H2O2/PBS for 15 min. Primary antibody incubation was performed
overnight (16 hr) at 4ÊC. Staining for GFP was preformed using an immunoperoxidase
technique (Vectastain Elite ABC kit, Vector Labs) and 3,3'-diaminobenzadine followed by
counterstaining with Meyer's hematoxylin. After counterstaining, samples were dehydrated through
ethanols and xylenes, mounted and cover slipped. Pdx1 IHC was performed using the Bond
RX autostainer (Leica Biosystems Inc.). For this, slides were baked at 65ÊC for 15 minutes and
the automated system performed dewaxing, rehydration, antigen retrieval, blocking, primary
antibody incubation, post primary antibody incubation, detection (DAB), and counterstaining
using Bond reagents (Leica). The following primary antibodies were used: rabbit anti-GFP
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(1:600, Abcam) and rabbit anti-Pdx1 (1:500, Abcam). Images were captured with either the
PerkinElmer's Vectra1 multispectral slide analysis system (GFP) or Zeiss Imager A.2 equipped
with a Zeiss Axiocam 512 color camera (Pdx1). All images were resized and formatted with
Adobe Photoshop CS5 software (Adobe Systems Incorporated).
To identify the Pdx1-FlpO knock-in allele, a genotyping strategy was designed including both
the 5' and 3' ends (Fig 1A). Primer sequences were: P1:50-GGAGAACTGTCAAAGCGATC-3’;
P4:5’TCGACTGTGCCTTCTAG TTG-3’. For the 5' end, the wild type allele amplified by primer pair
P1±P2 produces a PCR product of 576 bp whereas the Pdx1-FlpO allele amplified by primer
pair P1±P3 gives a 1100 bp product. For the 3' end, the wild type allele amplified by primer
pair P1±P2 produces a PCR product of 576 bp whereas the Pdx1-FlpO allele amplified by
primer pair P2±P4 gives a 399 bp product. All PCR reactions were performed using the
following conditions: 94ÊC for 1 min followed by 35 cycles of 94ÊC for 30 s, 55ÊC for 30 s, and 72ÊC 1
S1 Fig. Percentage of recombination in the pancreas and representative Pdx1
immunohistochemistry. (A) The percentage of recombination in the pancreas. The recombination was
quantified by percentage of GFP positive cells in three pancreatic epithelial lineages (ductal
cells, islet cells and acinar cells), N = 3 and (B) Representative Pdx1 immunohistochemistry
staining demonstrates Pdx1 expression in the pancreatic PanIN lesions (Black arrows) of
Pdx1FlpOki;FSF-KrasG12D/+;p53frt/+;FSFG–FP mice (scale bar: 50 μm).
S1 Table. Tumor and animal data.
The authors thank Dr. Tyler Jacks (Massachusetts Institute of Technology), Dr. David Kirsch
(Duke University) for providing transgenic animals; Dr. Mark Magnuson (Vanderbilt
University) for providing Pdx1CFP vector [
]; Dr. A. Francis Stewart for providing pCAGGs-FlpO
]; Dr. V. Coppola (Genetically Engineered Mouse Modeling facility of The Ohio State
University James Comprehensive Cancer Center) for helping mouse targeting; Shannon K.
Halloran, and Maokun Li for helping mouse housing and genotyping. This study was
supported by NIH PO1 CA097189 (M.C.O. and G.L.).
Conceptualization: Jinghai Wu, Gustavo Leone, Michael C. Ostrowski.
Data curation: Lianbo Yu, Blake E. Hildreth, III, Katie A. Thies.
Formal analysis: Jinghai Wu, Xin Liu.
Funding acquisition: Michael C. Ostrowski.
Investigation: Jinghai Wu, Xin Liu, Daniel J. Schilling, Michael C. Ostrowski.
Methodology: Jinghai Wu, Gustavo Leone, Michael C. Ostrowski.
11 / 13
Project administration: Jinghai Wu, Michael C. Ostrowski.
Resources: Soledad A. Fernandez, Michael C. Ostrowski.
Supervision: Michael C. Ostrowski.
Validation: Jinghai Wu, Xin Liu, Sunayana G. Nayak, Jason R. Pitarresi, Maria C. Cuitiño,
Soledad A. Fernandez.
Visualization: Xin Liu, Sunayana G. Nayak, Jason R. Pitarresi, Maria C. Cuitiño.
Writing ± original draft: Jinghai Wu, Xin Liu, Michael C. Ostrowski.
Writing ± review & editing: Jinghai Wu, Blake E. Hildreth, III, Katie A. Thies, Michael C.
12 / 13
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