115: Dual Recombinase Mouse Model to Dissect Cell Type-Specific Role of microRNA-21 in Pancreatic Cancer

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Presenter(s)

Katie Powell

Abstract or Description

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its highly aggressive cancer cells and extensive stroma reaction. This reactive stroma contains distinct tumor-restraining and tumor-promoting fibroblast subpopulations. We previously studied the effects of global loss of pro-fibrotic non-coding regulatory microRNA-21 (miR-21) in KRas-driven p53-deleted genetically engineered mouse models (GEMMs) of PDAC. The absence of tumor-restraining smooth muscle actin (SMA)–expressing myofibroblasts (myCAFs) and a massive infiltrate of immune cells were the most salient phenotypic features of global miR-21 loss. Stromal miR-21 activity was required for induction of tumor-restraining myofibroblasts in in-vivo isograft transplantation experiments. We hypothesize that cell-intrinsic activity of miR-21 is required for induction and maintenance of myCAFs. To test this hypothesis, we have generated a new dual recombinase mouse model to temporarily and spatially control the dose of miR-21 activity using the tamoxifen-inducible Flp/FRT system in combination with the Cre/LoxP system that activates the KRas-driven GEMMs. We describe the generation of a “flirted” Mir-21 allele flanked by FRT sites using easi-CRISPR technology and the characterization of mouse strains carrying one or two flirted alleles using molecular biology and histological analyses. We demonstrate the efficient recombination of flirted Mir-21 allele(s) in compounded strain with constitutive flippase (Rosa26-Flpe) or with tamoxifen-inducible flippase (Rosa26-FlpoER) after a single intraperitoneal injection of 75 mg/kg tamoxifen. We are crossing individual strains to produce a compounded strain with all the genetic elements required for this model. This dual recombinase system will be the first to conditionally delete the Mir-21 gene in myCAFs in well-established PDAC GEMMs. The genetic components of this dual recombinase system could be of general interest to the scientific community to mechanistically dissect the role of miR-21 in other cell types in PDAC by swapping the FlpoER driver strain or in myCAFs in other KRas-driven cancer types by swapping the Cre driver. Gained knowledge of the cell-intrinsic role of miR-21 in myCAFs and other cell types will be crucial to inform best strategies for pharmacological modulation of miR-21 activity for cancer interception and/or treatment in patients.