Chapter title |
Genetic Validation of Cell Proliferation via Ras-Independent Activation of the Raf/Mek/Erk Pathway.
|
---|---|
Chapter number | 20 |
Book title |
ERK Signaling
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-6424-6_20 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6422-2, 978-1-4939-6424-6
|
Authors |
Carmen G. Lechuga, Lucía Simón-Carrasco, Harrys K. C. Jacob, Matthias Drosten |
Editors |
Gerardo Jimenez |
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-Ras(lox) (H-Ras (-/-), N-Ras (-/-), K-Ras (lox/lox), RERT(ert/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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