Generation of a conditional Flpo/FRT mouse model expressing constitutively active TGF\u03b2 in fibroblasts

Ulrich Valcourt,Philippe Cassier,David F Vincent,Sylvie Martel,Adrien Bertrand-Chapel,Sophie Aires,Bastien Kaniewski,Nicolas Chuvin,Laurent Bartholin,Véronique Chauvet,Victoire Cardot-Ruffino,Roxane M Pommier,Cassandre Caligaris,Pierre Dubus,Stéphanie Sentis

doi:10.1038/s41598-020-60272-3

Abstract

Transforming growth factor (TGFβ) is a secreted factor, which accumulates in tissues during many physio- and pathological processes such as embryonic development, wound healing, fibrosis and cancer. In order to analyze the effects of increased microenvironmental TGFβ concentration in vivo, we developed a conditional transgenic mouse model (Flpo/Frt system) expressing bioactive TGFβ in fibroblasts, a cell population present in the microenvironment of almost all tissues. To achieve this, we created the genetically-engineered [Fsp1-Flpo; FSFTGFβCA] mouse model. The Fsp1-Flpo allele consists in the Flpo recombinase under the control of the Fsp1 (fibroblast-specific promoter 1) promoter. The FSFTGFβCA allele consists in a transgene encoding a constitutively active mutant form of TGFβ (TGFβCA) under the control of a Frt-STOP-Frt (FSF) cassette. The FSFTGFβCA allele was created to generate this model, and functionally validated by in vitro, ex vivo and in vivo techniques. [Fsp1-Flpo; FSFTGFβCA] animals do not present any obvious phenotype despite the correct expression of TGFβCA transgene in fibroblasts. This [Fsp1-Flpo; FSFTGFβCA] model is highly pertinent for future studies on the effect of increased microenvironmental bioactive TGFβ concentrations in mice bearing Cre-dependent genetic alterations in other compartments (epithelial or immune compartments for instance). These dual recombinase system (DRS) approaches will enable scientists to study uncoupled spatiotemporal regulation of different genetic alterations within the same mouse, thus better replicating the complexity of human diseases.

Highlights

TGFβ is the archetype of the transforming growth factor superfamily[1]
In the presence of a recombinase of the Flp family, the transcriptional STOP signal can be excised and the TGFβCA transgene expressed under the control of the ubiquitous cytomegalovirus (CMV) early enhancer/chicken β-actin promoter (CAG)
In order to validate the conditional expression of the TGFβCA transgene ex vivo, we prepared ear skin primary fibroblasts from [FSFTGFβCA] mice

Summary

Introduction

TGFβ (transforming growth factor beta) is the archetype of the transforming growth factor superfamily[1]. We constructed a recombinant targeting vector expressing the FSFTGFβCA transgene and encoding a Flp-conditional (FSF, Frt-STOP-Frt cassette) constitutively active (CA) TGFβ1 mutant (TGFβCA) (Fig. 1a). In order to validate the conditional expression of the TGFβCA transgene ex vivo, we prepared ear skin primary fibroblasts from [FSFTGFβCA] mice.

Results

Conclusion