title: Genetic Validation of Cell Proliferation via Ras-Independent Activation of the Raf/Mek/Erk Pathway.
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.
authors.affiliation: Molecular Oncology Programme,, Centro Nacional de Investigaciones Oncológicas (CNIO), Melchor Fernández Almagro 3, Madrid, 28029, Spain.
authors.forename: Carmen G
authors.initials: CG
authors.lastname: Lechuga
authors.affiliation: Molecular Oncology Programme,, Centro Nacional de Investigaciones Oncológicas (CNIO), Melchor Fernández Almagro 3, Madrid, 28029, Spain.
authors.forename: Lucía
authors.initials: L
authors.lastname: Simón-Carrasco
authors.affiliation: Molecular Oncology Programme,, Centro Nacional de Investigaciones Oncológicas (CNIO), Melchor Fernández Almagro 3, Madrid, 28029, Spain.
authors.forename: Harrys K C
authors.initials: HK
authors.lastname: Jacob
authors.affiliation: Molecular Oncology Programme,, Centro Nacional de Investigaciones Oncológicas (CNIO), Melchor Fernández Almagro 3, Madrid, 28029, Spain. mdrosten@cnio.es.
authors.forename: Matthias
authors.initials: M
authors.lastname: Drosten
mesh_terms: D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D055786:Gene Knockout Techniques; D056726:Genetic Association Studies; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D012190:Retroviridae / Q000235:genetics; D015398:Signal Transduction; D014161:Transduction, Genetic; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins
keywords: Cell proliferation; Colony formation assay; Raf/Mek/Erk pathway; Ras independent; Ras signaling