Development of a new flippase-dependent mouse model for red fluorescence-based isolation of KrasG12D
Dusan Hrckulak1, Michaela Krausova1, Jakub Onhajzer1
1Laboratory of Cell and Developmental Biology, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, 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 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 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
We developed a novel RedRas knock-in model for studying oncogenic Kras. This model allows visualization and isolation of Kras-driven tumor cells, aiding cancer research.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Proto-oncogene KRAS (Kras) is a key target in cancer research.
- Existing mouse models struggle with isolating Kras-expressing tumor cells.
Purpose of the Study:
- To present a new knock-in model, KrasRR (RedRas), for studying oncogenic Kras.
- To enable visualization and isolation of cells expressing mutant KrasG12D.
Main Methods:
- Developed a KrasG12D knock-in allele (KrasRR) linked to tdTomato fluorescence.
- Utilized Flp recombinase for conditional KrasG12D activation.
- Validated the model in vitro (fibroblasts, organoids) and in vivo (lung, colon).
Main Results:
- KrasRR allele activation triggers lung adenomas and colon adenoma progression.
- tdTomato fluorescence allows for visualization and isolation of recombined cells.
- KrasG12D drives organoid growth independently of EGF signaling and is inhibited by MRTX1133.
Conclusions:
- The RedRas model provides a powerful tool for studying Kras oncogene function in cancer.
- This model facilitates the isolation and analysis of tumor cells in vivo.
- KrasG12D-driven tumorigenesis can be targeted by specific inhibitors like MRTX1133.
