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. 2019 Nov 20;9:17220. doi: 10.1038/s41598-019-53572-w

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Immortalized MEFs expressing endogenous Kras^G12D are transformed by mutation of Trp53. (A) Kras^LSL-G12D MEFs (MEF-K), Kras^LSL-G12D; Rosa26^{LSL-Cas9-EGFP/+} MEFs (MEF-KC), Rosa26^{LSL-Cas9-EGFP/+} MEFs (MEF-C) were infected with adenovirus expressing Cre (Ad-Cre) and then genotyped using PCR to confirm recombination of the floxed STOP cassette (LoxP). (B) Ad-Cre infected MEFs (MEF-LoxP-K, MEF-LoxP-KC, MEF-LoxP-C) were cultured for more than 10 passages and were stained with reagents for the β-Galactosidase senescence assay. (C) Immortalized MEF-LoxP-KC cells were transduced with lentivirus expressing either negative control sgRNA (lenti-Neg) or a sgRNA targeting Trp53 (lenti-Trp53), and then seeded in soft agar or allografted in nude mice. MEF-LoxP-KC cells infected with Trp53 sgRNA resulted in anchorage-independent growth in the soft agar assay and tumor formation in nude mice allografts (n = 2 or 3). The result is representative of at least two independent experiments. Scale bars = 100 µm.