Development of a new flippase-dependent mouse model for red fluorescence-based isolation of KRASG12D
Dusan Hrckulak1, Jakub Onhajzer1, Michaela Krausova1,2
1Laboratory of Cell and Developmental Biology, Institute of Molecular Genetics of the Czech Academy of Sciences, Videnska 1083, 142 20, Prague 4, Czech Republic.
Abstract:
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 KRASG12D that enables the "activation" of KRASG12D 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 KrasRR) 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 KrasRR 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 KRASG12D production enables intestinal organoid growth independent of epidermal growth factor (EGF) signaling and that the KRASG12D function is effectively suppressed by specific inhibitor MRTX1133.
Insights
Researchers developed a new RedRas mouse model for studying KRAS oncogenes. This model allows visualization and isolation of KRASG12D-expressing cancer cells, aiding tumor analysis and therapeutic development.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Proto-oncogene KRAS (GTPase) is a critical target in cancer research.
- Existing mouse models face challenges in isolating and analyzing KRAS-driven tumor cells.
- Targeted analysis of oncogenic KRAS signaling is crucial for understanding tumorigenesis.
Purpose of the Study:
- To develop a novel knock-in mouse model for studying oncogenic KRASG12D.
- To enable visualization and isolation of cells expressing mutant KRAS.
- To investigate KRASG12D-driven tumorigenesis in lung and colon epithelium.
Main Methods:
- Created a RedRas (KrasRR) knock-in allele enabling KRASG12D and tdTomato co-expression upon Flp-mediated recombination.
- Validated the KrasRR allele in vitro using mouse embryonic fibroblasts and organoids.
- Assessed KrasRR functionality in vivo within lung and colon epithelium models.
Main Results:
- Recombination of the KrasRR allele induced lung adenoma formation and promoted colon adenoma progression.
- tdTomato fluorescence allowed for visualization and isolation of recombined cells.
- KRASG12D expression drove intestinal organoid growth independently of EGF signaling and was inhibited by MRTX1133.
Conclusions:
- The RedRas (KrasRR) model provides a powerful tool for studying oncogenic KRAS.
- This model facilitates the isolation and analysis of KRAS-driven tumor cells.
- The findings support the development of targeted KRAS inhibitors like MRTX1133.
