BRIEF COMMUNICATIONS NATURE METHODS | ADVANCE ONLINE PUBLICATION | 1 Ras proteins are aberrantly activated as a result of mutations in many cancers 2,3 . Activated Ras has high-affinity interactions with several of its downstream effector molecules, and this proc- ess is implicated in tumorigenesis 4,5 . The best-characterized Ras effector function is activation of the Raf–mitogen activated protein kinase (Ras-MAPK) cascade. Initially it was thought that the oncogenic functions of Ras are mediated through Raf 6 ; however, other Ras effector proteins were later also implicated. Among these proteins, the Ral guanine nucleotide exchange factor (RALGEF) and phosphatitylinositol-3-kinase (PI3K) pathways have attracted particular attention, and additional studies have questioned the original dogma of Raf being the most important mediator of Ras in oncogenesis 5 . To dissect the requirement of Ras downstream effector pathways in tumorigenesis, we generated a Ras effector (RasE) Multi-Hit transgenic mouse. For ease of use, we established a recombineering method for the rapid assembly of complex Multi-Hit bacterial arti- ficial chromosome (BAC) constructs (Supplementary Fig. 2) 7,8 . The RasE Multi-Hit BAC contained expression units for alleles of Hras with the G12V substitution (HrasV12) and with an additional T35S substitution (Hras V12S35 ), an additional E37G substitution (Hras V12G37 ) or an additional Y40C substitution (Hras V12C40 . m ) (Fig. 1a). These oncogenic Hras mutant alleles carry individual mutations in the Ras effector domain and are surrogates for Ras-induced selective activation of the MAPK (Hras V12S35 ), RALGEF (Hras V12G37 ) and PI3K (Hras V12C40 ) downstream effec- tor pathways 9 . For simplicity, we indicate the Hras V12S35 (MAPK), Hras V12G37 (RALGEF) and Hras V12C40 (PI3K) alleles with letters M, R and P, respectively. We addressed the issue of a possible nonstochastic Cre- and loxP-mediated bias in Ras effector activa- tion in embryonic stem cells with single integration of the RasE Multi-Hit BAC (ES RasE cells). We observed subtle differences in allele activation but no major predominance of certain activation events (Supplementary Fig. 3). We used the RasE Multi-Hit BAC for the generation of RasE transgenic mice (Supplementary Fig. 4a). RasE mice did not develop tumors within the time monitored (up to 2 years of age). According to the Multi-Hit principle, Cre induction in RasE mice should give rise to eight genotypically different cell types with differential activation of Ras effector pathways (Fig. 1b). We first crossed RasE mice with inducible Rosa26CreER T2 mice (to gene- rate RasE CreER mice) and found activation of the M, R and P alleles after injecting these mice with tamoxifen (Supplementary Fig. 4b). Consistently, Ras protein concentrations increased when compared Q2 Q2 A mouse model to identify cooperating signaling pathways in cancer Monica Musteanu 1,2,11 , Leander Blaas 1,11 , Rainer Zenz 1 , Jasmin Svinka 1 , Thomas Hoffmann 1 , Beatrice Grabner 1 , Daniel Schramek 3 , Hans-Peter Kantner 1 , Mathias Müller 4 , Thomas Kolbe 5,6 , Thomas Rülicke 5 , Richard Moriggl 1 , Lukas Kenner 1,7 , Dagmar Stoiber 1,8 , Josef Martin Penninger 3 , Helmut Popper 9 , Emilio Casanova 1,11 & Robert Eferl 1,10,11 . m We here establish a mouse cancer model called Multi-Hit that allows for the evaluation of oncogene cooperativities in tumor development. The model is based on the stochastic expression of oncogene combinations (‘hits’) that are mediated by Cre in a given tissue. Cells with cooperating hits are positively selected and give rise to tumors. We used this approach to evaluate the requirement of Ras downstream effector pathways in tumorigenesis. Tumors arise from selected cells that have accumulated mutations (hits) in proto-oncogenes or tumor-suppressor genes leading to cel- lular characteristics known as hallmarks of cancer 1 . The underlying positive selection leads to tumor cells that have been transformed by potently cooperating oncogenic mutations. We developed a Multi- Hit mouse model that takes into account positive selection of trans- formed cells with cooperating mutations (Fig. 1a,b). This approach uses Cre-mediated stochastic activation of expression modules that are flanked by heterotypic loxP sites. After transient induction of Cre, expression modules flip until Cre activity ceases. They then remain in either an on or an off orientation. Therefore, Cre induction in a Multi-Hit mouse results in the stochastic activation of expression modules, creating eight genotypically different cell types. Cells with tumor-promoting gene expression patterns have a selective advan- tage to give rise to tumors (Fig. 1b). Moreover, combined approaches could use additional hits (for example, treatment of Multi-Hit mice with mutagens before stochastic oncogene activation or deletion of tumor-suppressor genes; Supplementary Fig. 1). Q1 Q1 1 Ludwig Boltzmann Institute for Cancer Research, Vienna, Austria. 2 Spanish National Cancer Research Centre (CNIO), Madrid, Spain. 3 Institute of Molecular Biotechnology, Vienna, Austria. 4 Institute of Animal Breeding and Genetics, University of Veterinary Medicine, Vienna, Austria. 5 Institute of Laboratory Animal Science and Biomodels Austria, University of Veterinary Medicine, Vienna, Austria. 6 Department for Agrobiotechnology, Biotechnology in Animal Production, University of Natural Resources and Applied Life Sciences, Tulln, Austria. 7 Clinical Institute of Pathology, Medical University of Vienna, Vienna, Austria. 8 Center of Physiology and Pharmacology, Institute of Pharmacology, Medical University of Vienna, Vienna, Austria. 9 Institute of Pathology, Medical University of Graz, Graz, Austria. 10 Institute for Cancer Research (ICR), Comprehensive Cancer Center (CCC), Medical University of Vienna, Vienna, Austria. 11 These authors contributed equally to this work. Correspondence should be addressed to R.E. (robert.eferl@meduniwien.ac.at). RECEIVED 29 MARCH; ACCEPTED 10 JULY; PUBLISHED ONLINE XX XX 2012; DOI:10.1038/NMETH.2130