Abstract
RAS proteins are key players in multiple cellular processes. To study the role of RAS proteins individually or in combination, we have developed MEFs that can be rendered RASless, i.e., devoid of all endogenous RAS isoforms. These cells have significantly contributed to our understanding of the requirements for RAS functions in cell proliferation as well as their implications in diverse cellular processes. Here, we describe methods using RASless MEFs to study RAS-dependent cellular activities with special emphasis on proliferation. We provide the details to identify inducers of RAS-independent proliferation in colony assays. We recommend following these stringent guidelines to avoid false-positive results. Moreover, this protocol can be adapted to generate RASless MEFs ectopically expressing RAS variants to interrogate their function in the absence of endogenous RAS isoforms or to perform experiments in the absence of RAS. Finally, we also describe protocols to generate and use RASless MEFs for cell cycle analyses using the FUCCI cell cycle indicator.
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Proliferation of KrasloxFUCCI cells stably expressing the monomeric version of Kusabira Orange 2 (mKO2)/human chromatin licensing and DNA replication factor 1 (hCdt1) (red) and the monomeric version of Azami Green (mAG)/human Geminin (hGeminin) (green). The video was generated by taking one image every 10 min for 50 h (10 frames/s) (MP4 2384 kb)
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Lechuga, C.G., Salmón, M., Paniagua, G., Guerra, C., Barbacid, M., Drosten, M. (2021). RASless MEFs as a Tool to Study RAS-Dependent and RAS-Independent Functions. In: Rubio, I., Prior, I. (eds) Ras Activity and Signaling. Methods in Molecular Biology, vol 2262. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1190-6_21
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DOI: https://doi.org/10.1007/978-1-0716-1190-6_21
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