Abstract
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.
Highlights
Conditional gene knockout is a powerful tool to study the role of individual genes in living organisms
The results presented demonstrate the exclusive expression of FlpO in pancreas-duodenum homeobox 1 (Pdx1) expressing tissues in our knock-in PdxFlpO mouse model
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
Summary
Conditional gene knockout is a powerful tool to study the role of individual genes in living organisms. 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. 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. Conditional gene knockout mice are often used as models of human diseases It has tremendously increased our ability to study complex human diseases in specific developmental stages and tissues through spatial or temporal gene inactivation via DNA excision. 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 distinct recombinases
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