将cre loxp与crispr cas9结合的方法

The EMBO Journal Vol 35 | No 2 | 2016 143
Genetic analyses demonstrated that Raf isoforms possess unique and overlapping functions. B-Raf is required for maximum ERK activation and for placental development as it is illustrated by the embryonic lethality of B-Raf-deficient mice (Wojnowski et al, 2000; Brummer et al, 2002; Galabova-Kovacs et al, 2006, 2008; Zhong et al, 2007). B-Raf displays the most potent transforming activity among the three isoforms and is often activated by somatic alterations in cancer (Pritchard et al, 1995; Holderfield et al, 2014). Consequently, B-Raf appears as an attractive therapeutic target and inhibitors such as vemurafenib yield unprecedented response rates
1 Faculty of Medicine, Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University (ALU), Freiburg, Germany 2 Centre for Biological Systems Analysis ZBSA, ALU, Freiburg, Germany 3 Spemann Graduate School for Biology and Medicine, ALU, Freiburg, Germany 4 Faculty of Biology, ALU, Freiburg, Germany 5 Department of Hematology and Oncology, University Medical Center, ALU, Freiburg, Germany 6 Centre for Biological Signalling Studies BIOSS, ALU, Freiburg, Germany 7 Department of Molecular Immunology, Faculty of Biology, University of Freiburg, Freiburg, Germany 8 Comprehensive Cancer Centre, Freiburg, Germany 9 German Consortium for Translational Cancer Research DKTK, Standort பைடு நூலகம்reiburg, Germany 10 Center for Chronic Immunodeficiency CCI, University Medical Center, Freiburg, Germany 11 Institute of Immunology, University Hospital Ulm, Ulm, Germany
Published online: December 10, 2015
Article
Activation loop phosphorylation regulates B-Raf in vivo and transformation by B-Raf mutants
Martin Köhler1,2,3,4,†, Michael Röring1,2,3,4,†, Björn Schorch2,3,4, Katharina Heilmann2,4, Natalie Stickel3,4,5, Gina J Fiala3,4,6,7, Lisa C Schmitt1,2,4, Sandra Braun1,2,6, Sophia Ehrenfeld1,4, Franziska M Uhl1,4, Thorsten Kaltenbacher1,4, Florian Weinberg1,2,4, Sebastian Herzog2,6,7, Robert Zeiser5,6,8,9, Wolfgang W Schamel6,7,10, Hassan Jumaa6,7,11 & Tilman Brummer1,2,4,6,8,9,*
See also: A Varga & M Baccarini (January 2016)
The Ras/Raf/MEK/ERK pathway plays a pivotal role in controlling proliferation, survival, and differentiation. As this pathway is often deregulated in various diseases, in particular cancer and RASopathies, its components are pursued as targets for pharmacological intervention (Holderfield et al, 2014; Samatar & Poulikakos, 2014). Raf kinases represent a particularly important node as they are subject to a complex and tight regulation (Cseh et al, 2014). The Raf family comprises A-Raf, B-Raf, and Raf-1 in vertebrates and single raf genes in invertebrates such as D-Raf and LIN-45 in Drosophila and Caenorhabditis, respectively. Distant relatives are the KSR proteins, which promote MEK activation as scaffolds, allosteric Raf activators, and potentially by their own kinase activity (Brennan et al, 2011). As described below, the presence of three Raf and two KSR isoforms allows fine-tuning of Raf activity by forming homoand heterodimers with distinct signaling potential (Cseh et al, 2014; Mooz et al, 2014). For example, B-Raf/Raf-1 heterodimers represent the most potent MEK activator (Rushworth et al, 2006; Freeman et al, 2013).
Keywords BRAF fusion; Cre/loxP system; gene targeting; MAPK pathway; Ras Subject Categories Cancer; Signal Transduction DOI 10.15252/embj.201592097 | Received 19 May 2015 | Revised 26 October 2015 | Accepted 28 October 2015 | Published online 10 December 2015 The EMBO Journal (2016) 35: 143–161
*Corresponding author. Tel: +49 7612 039610; Fax: +49 7612 039602; E-mail: tilman.brummer@zbsa.de †These authors contributed equally to this work
ª 2015 The Authors
Abstract
Introduction
Despite being mutated in cancer and RASopathies, the role of the activation segment (AS) has not been addressed for B-Raf signaling in vivo. Here, we generated a conditional knock-in mouse allowing the expression of the B-RafAVKA mutant in which the AS phosphoacceptor sites T599 and S602 are replaced by alanine residues. Surprisingly, despite producing a kinase-impaired protein, the Braf AVKA allele does not phenocopy the lethality of Braf-knockout or paradoxically acting knock-in alleles. However, BrafAVKA mice display abnormalities in the hematopoietic system, a distinct facial morphology, reduced ERK pathway activity in the brain, and an abnormal gait. This phenotype suggests that maximum B-Raf activity is required for the proper development, function, and maintenance of certain cell populations. By establishing conditional murine embryonic fibroblast cultures, we further show that MEK/ ERK phosphorylation and the immediate early gene response toward growth factors are impaired in the presence of B-RafAVKA. Importantly, alanine substitution of T599/S602 impairs the transformation potential of oncogenic non-V600E B-Raf mutants and a fusion protein, suggesting that blocking their phosphorylation could represent an alternative strategy to ATP-competitive inhibitors.