title: P2A-Fluorophore Tagging of BRAF Tightly Links Expression to Fluorescence In Vivo.
abstract: The Braf proto-oncogene is a key component of the mitogen-activated protein kinase signaling cascade and is a critical regulator of both normal development and tumorigenesis in a variety of tissues. In order to elucidate BRAF's differing roles in varying cell types, it is important to understand both the pattern and timing of BRAF expression. Here we report the production of a mouse model that links the expression of Braf with the bright red fluorescent protein, tdTomato. We have utilized a P2A knock-in strategy, ensuring that BRAF and the fluorophore are expressed from the same endogenous promoter and from the same bicistronic mRNA transcript. This mouse model (BrafTOM) shows bright red fluorescence in organs and cell types known to be sensitive to BRAF perturbation. We further show that on a cell-by-cell basis, fluorescence correlates with BRAF protein levels. Finally, we extend the utility of this mouse by demonstrating that the remnant P2A fragment attached to BRAF acts as a suitable epitope for immunoprecipitation and biochemical characterization of BRAF in vivo.
authors.affiliation: Helen Diller Family Comprehensive Cancer, University of California San Francisco, San Francisco, CA, United States of America.|Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, United States of America.
authors.forename: J Edward
authors.identifier: http://orcid.org/0000-0003-1798-3210
authors.initials: JE
authors.lastname: van Veen
authors.affiliation: Helen Diller Family Comprehensive Cancer, University of California San Francisco, San Francisco, CA, United States of America.|Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, United States of America.
authors.forename: Daphne R
authors.initials: DR
authors.lastname: Pringle
authors.affiliation: Helen Diller Family Comprehensive Cancer, University of California San Francisco, San Francisco, CA, United States of America.|Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, United States of America.
authors.forename: Martin
authors.initials: M
authors.lastname: McMahon
mesh_terms: D000818:Animals; D005434:Flow Cytometry / Q000379:methods; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D061848:Optical Imaging / Q000379:methods*; D000072417:Protein Domains; D048493:Proto-Oncogene Proteins B-raf / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D048493:Proto-Oncogene Proteins B-raf; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein; C482120:Braf protein, mouse