Development of a new flippase-dependent mouse model for red fluorescence-based isolation of KRASG12D oncogene-expressing tumor cells.

Proto-oncogene KRAS, GTPase (KRAS) is one of the most intensively studied oncogenes in cancer research. Although several mouse models allow for regulated expression of mutant KRAS, selective isolation and analysis of transforming or tumor cells that produce the KRAS oncogene remains a challenge. In our study, we present a knock-in model of oncogenic variant KRAS^G12D that enables the "activation" of KRAS^G12D expression together with production of red fluorescent protein tdTomato. Both proteins are expressed from the endogenous Kras locus after recombination of a transcriptional stop box in the genomic DNA by the enzyme flippase (Flp). We have demonstrated the functionality of the allele termed RedRas (abbreviated Kras^RR) under in vitro conditions with mouse embryonic fibroblasts and organoids and in vivo in the lung and colon epithelium. After recombination with adenoviral vectors carrying the Flp gene, the Kras^RR allele itself triggers formation of lung adenomas. In the colon epithelium, it causes the progression of adenomas that are triggered by the loss of tumor suppressor adenomatous polyposis coli (APC). Importantly, cells in which recombination has successfully occurred can be visualized and isolated using the fluorescence emitted by tdTomato. Furthermore, we show that KRAS^G12D production enables intestinal organoid growth independent of epidermal growth factor (EGF) signaling and that the KRAS^G12D function is effectively suppressed by specific inhibitor MRTX1133.