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ERK Signaling pp 269-276 | Cite as

Genetic Validation of Cell Proliferation via Ras-Independent Activation of the Raf/Mek/Erk Pathway

  • Carmen G. Lechuga
  • Lucía Simón-Carrasco
  • Harrys K. C. Jacob
  • Matthias DrostenEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1487)

Abstract

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.

Key words

Ras signaling Cell proliferation Ras independent Colony formation assay Raf/Mek/Erk pathway 

Notes

Acknowledgments

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).

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Carmen G. Lechuga
    • 1
  • Lucía Simón-Carrasco
    • 1
  • Harrys K. C. Jacob
    • 1
  • Matthias Drosten
    • 1
    Email author
  1. 1.Molecular Oncology Programme,Centro Nacional de Investigaciones Oncológicas (CNIO)MadridSpain

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