Generation of an Fsp1 (fibroblast‐specific protein 1)‐Flpo transgenic mouse strain

Summary Recombination systems represent a major breakthrough in the field of genetic model engineering. The Flp recombinases (Flp, Flpe, and Flpo) bind and cleave DNA Frt sites. We created a transgenic mouse strain ([Fsp1‐Flpo]) expressing the Flpo recombinase in fibroblasts. This strain was obtained by random insertion inside mouse zygotes after pronuclear injection. Flpo expression was placed under the control of the promoter of Fsp1 (fibroblast‐specific protein 1) gene, whose expression starts after gastrulation at Day 8.5 in cells of mesenchymal origin. We verified the correct expression and function of the Flpo enzyme by several ex vivo and in vivo approaches. The [Fsp1‐Flpo] strain represents a genuine tool to further target the recombination of transgenes with Frt sites specifically in cells of mesenchymal origin or with a fibroblastic phenotype.

Recently, a light-sensitive Flpo recombinase was developed as a nontoxic and noninvasive alternative to tamoxifen (Flpo recombinase active heterodimers can form after LED light illumination of a specific zone [Jung et al., 2019]). Despite these advances, the development of organ-or cell-specific "Flp" mice remains limited, hindering the possibility of taking advantage of the Flp/Frt recombination system in vivo.
Considering the crucial role of fibroblasts in the microenvironment, we created in the present study an original mouse model expressing the Flpo recombinase in the fibroblastic compartment ([Fsp1-Flpo] mouse strain). We chose the Flp/Frt approach with the final goal in the future of combining this system with the Cre/lox system in dual recombinase systems (DRS), which consist in combining two recombinase systems within the same mouse (e.g., Cre/Lox and Flp/Frt).

| RESULTS AND DISCUSSION
The Fsp1 promoter consists in a~3 kbp genomic DNA fragment that drives gene expression in fibroblasts (Bhowmick et al., 2004;Iwano et al., 2002;Okada et al., 1998;Strutz et al., 1995). The Flpo transgene is a mouse codon-optimized Flp (Flpo) site-specific recombinase (SSR), which recombines DNA Frt-sites (Raymond & Soriano, 2007). were isolated and cultured as follows: mouse ears were rinsed with 70% ethanol and samples of a few square-millimeters were harvested. Primary ear fibroblasts were isolated using mechanical dilaceration, followed by enzymatic dissociation (600 μl of a mix of collagenase D (4 mg ml −1 , COLLD-RO Roche)/Dispase II (4 mg ml −1 , Roche) in RPMI medium) at 37 C for 1 hr. The reaction was stopped by adding 5 ml of complete medium (Dulbecco's Modified Eagle's Medium, supplemented with 0.03% L-glutamine and containing 10% fetal bovine serum, a mix of 100 U ml −1 penicillin and 100 μg ml −1 streptomycin sulfate) and cells were then incubated at 37 C overnight. The following day, after filtration through a 100 μm pore cell strainer, cells were pelleted and reseeded in complete medium (Dulbecco's Modified Eagle's Medium, supplemented with 0.03% L-glutamine and containing 10% fetal bovine serum, a mix of 100 U ml −1 penicillin and 100 μg ml −1 streptomycin sulfate, 1% MEM nonessential amino acids and 50 μM β-mercaptoethanol). Medium was changed after 48 hr. All cells were propagated at 37 C under 5% (v/v) CO 2 atmosphere.
Sorted skin cells were prepared as follows: mice were sacrificed and shaved, and a piece of skin from the back of the animals was harvested and fat was removed. Five milliliter of digestion medium (RPMI1640, 20% FBS, 1% PS, 1% Hepes, 1% Glutamine) was added to the skin in a petri dish. The skin was dilacerated using scissors and a cutter. Another 5 ml of digestion medium was added to help harvest the mixture. The mixture was then transferred to 50 ml tubes under agitation with a magnetic bar. One milliliter of collagenase type IA

| Mice
The [Fsp1-Flpo] mouse strain will be available to the research community upon acceptance of the manuscript.
The [ FSF hPLAP] strain was previously described (Awatramani et al., 2001) and was obtained from Charles River Laboratories ((Gt(ROSA) Fifty micro liter of neutralization buffer (40 mM Tris-HCl) was then added. DNA extraction and PCR were performed as previously described (Vincent et al., 2010) using Taq DNA polymerase (Life Technologies) and primers cited in Table 1

| Reverse-transcription PCR
First-strand cDNAs were prepared using 250 ng of RNA and Super-Script II Reverse Transcriptase in the presence of random primers (Invitrogen) (Vincent et al., 2009). Semi-quantitative PCR on cDNA was performed as previously described (Pommier et al., 2012)
The separated proteins were transferred onto PVDF membranes (Millipore) by electroblotting. Western blots were visualized using the ECL Western detection system (GE Healthcare). Images were captured using a ChemiDoc Biorad MP. We used human placental alkaline phosphatase primary antibody, rabbit monoclonal 1:500 (Abcam ab16695 [SP15]), GAPDH, mouse monoclonal primary antibody, 1:1,000 (Abcam ab8245) and the secondary antibodies were Alkaline phosphatase (AP) staining was performed as follows: 10 μm-thick sections of frozen tissues were cut using the Cryostat Sections were subsequently placed in AP staining solution (AP detection buffer with 0.8 mg/ml nitroblue tetrazolium (NBT), 0.1 mg/ml 5-bromo-4-chloro-3-indolyl phosphate dimethylformamide (BCIP)) at room temperature for 2-6 hr. Once color development was complete, sections were washed in PBS 1X.

| Statistical analysis
All graphs display one representative experiment performed three times. Prism 7.0 (Graphpad) was used for statistics and to create graphs. The error bars represent the standard deviation (SD) from technical duplicates.  English language editing of the manuscript.

CONFLICT OF INTEREST
The authors declare no conflict of interest.