Signaling transmitted by the Ras family of small GTPases (H-, N-, and K-Ras) is essential for proliferation of mouse embryonic fibroblasts (MEFs). However, constitutive activation of the downstream Raf/Mek/Erk pathway can bypass the requirement for Ras proteins and allow cells to proliferate in the absence of the three Ras isoforms. Here we describe a protocol for a colony formation assay that permits evaluating the role of candidate proteins that are positive or negative regulators of cell proliferation mediated via Ras-independent Raf/Mek/Erk pathway activation. K-Raslox (H-Ras–/–, N-Ras–/–, K-Raslox/lox, RERTert/ert) MEFs are infected with retro- or lentiviral vectors expressing wild-type or constitutively activated candidate cDNAs, shRNAs, or sgRNAs in combination with Cas9 to ascertain the possibility of candidate proteins to function either as an activator or inhibitor of Ras-independent Raf/Mek/Erk activation. These cells are then seeded in the absence or presence of 4-Hydroxytamoxifen (4-OHT), which activates the resident CreERT2 alleles resulting in elimination of the conditional K-Ras alleles and ultimately generating Rasless cells. Colony formation in the presence of 4-OHT indicates cell proliferation via Ras-independent Raf/Mek/Erk activation.
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This work was supported by grants from the EU-Framework Program (HEALTH-F2-2010-259770/LUNGTARGET and HEALTH-2010-260791/EUROCANPLATFORM), Spanish Ministry of Economy (SAF2011-30173) and Autonomous Community of Madrid (S2011/BDM-2470/ONCOCYCLE).
Esteban LM, Vicario-Abejón C, Fernández-Salguero P et al (2001) Targeted genomic disruption of H-ras and N-ras, individually or in combination, reveals the dispensability of both loci for mouse growth and development. Mol Cell Biol 21:1444–1452CrossRefPubMedPubMedCentralGoogle Scholar
Koera K, Nakamura K, Nakao K et al (1997) K-Ras is essential for the development of the mouse embryo. Oncogene 15:1151–159CrossRefPubMedGoogle Scholar
Potenza N, Vecchione C, Notte A et al (2005) Replacement of K-Ras with H-Ras supports normal embryonic development despite inducing cardiovascular pathology in adult mice. EMBO Rep 6:432–437CrossRefPubMedPubMedCentralGoogle Scholar
Stokoe D, Macdonald SG, Cadwallader K et al (1994) Activation of Raf as a result of recruitment to the plasma membrane. Science 264:1463–1467CrossRefPubMedGoogle Scholar
Drosten M, Sum EY, Lechuga CG et al (2014) Loss of p53 induces cell proliferation via Ras-independent activation of the Raf/Mek/Erk pathway. Proc Natl Acad Sci USA 111:15155–15160CrossRefPubMedPubMedCentralGoogle Scholar