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PLoS One. 2016 Jun 27;11(6):e0157661. doi: 10.1371/journal.pone.0157661. eCollection 2016.

P2A-Fluorophore Tagging of BRAF Tightly Links Expression to Fluorescence In Vivo.

Author information

1
Helen Diller Family Comprehensive Cancer, University of California San Francisco, San Francisco, CA, United States of America.
2
Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, United States of America.

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.

PMID:
27348307
PMCID:
PMC4922626
DOI:
10.1371/journal.pone.0157661
[Indexed for MEDLINE]
Free PMC Article

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