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.

Mutationally-activated PI3’-kinase-α promotes de-differentiation of lung tumors initiated by the BRAFV600E oncoprotein kinase

“…Because BRAF is expressed in ES cells, we reasoned that modification of the normal allele would lead to ES cells with constitutive tdTomato-CAAX expression. Indeed, ~50% of PCR positive ES cell clones displayed constitutive membrane associated red-fluorescence and were used to generate Braf TOM mice, in which tdTomato serves as a marker for any cells expressing normal BRAF (van Veen et al, 2016). By contrast, homologous recombination of the targeting vector into the Braf CA allele should give rise to ES cells that do not express tdTomato due to the strong transcriptional termination signal.…”

“…While this is acceptable for some applications, it adds confounding noise that may be amplified under conditions when tumor cells have been specifically depleted by the application of pathway targeted or immunomodulatory therapy that has negligible effect on normal cells. Our approach avoids this noise entirely and results in a level of fluorescence that is directly correlated with expression of BRAF V600E on a cell-by-cell basis (van Veen et al, 2016). Importantly, our approach is especially compatible with the emerging technology of single cell RNA sequencing (scRNA-Seq), in which it is desirable to isolate single BRAF V600E oncoprotein kinase expressing cells for analysis.…”

Transposon Mutagenesis Reveals RBMS3 as a Promoter of Malignant Progression of BRAFV600E-Driven Lung Tumorigenesis

“…Genetically engineered mouse (GEM) models of human cancer provide a powerful platform to test hypotheses regarding tumor initiation, cancer progression and maintenance. Braf CA , as well as Braf CAT mice were engineered to express normal BRAF prior to CRE-mediated recombination after which BRAF V637E (analogous to human BRAF V600E , the nomenclature that we will use to describe this model hereafter) is expressed in cells in a temporally and spatially restricted manner (Dankort et al 2007; van Veen et al 2016; van Veen et al 2019). The Braf CAT mice were further adapted to express the tdTomato flourescent reporter upon additional of CRE as well.…”