STOCK Gt(ROSA)26Sor^{tm65.2(CAG-tdTomato)Hze} Pdgfrb^{tm1.1(flpo)Leol}/LeolJ

Stock number: 038629
Product name: Pdgfrb^Flp;fSf-Tomato
Strain synonyms: bR-Flp;fSf-Tomato
Research areas: Research Tools

Description

Pdgfrb^Flp;fSf-Tomato mice have a floxed cassette containing both a cDNA encoding the Pdgfrb gene and a flpo sequence replacing exons 2-4 of the endogenous Pdgfrb gene. Mice also contain a conditional tdTomato fluorescent FLP reporter allele, Ai65F or fSf-Tomato. Cre recombination leads to the knock-out of Pdgfrb expression and Flp/FRT-mediated intersectional lineage tracing of cre-expressing cells and their progeny.

Detailed description

Pdgfrb^Flp;fSf-Tomato mice have a floxed cassette containing both a cDNA encoding the platelet derived growth factor receptor, beta (Pdgfrb) gene and a flpo sequence replacing exons 2-4 of the endogenous Pdgfrb gene. Mice also contain a conditional tdTomato fluorescent FLP reporter allele, Ai65F or fSf-Tomato. Pdgfrb is expressed in most mural cells and is involved in blood vessel development, and the migration and recruitment of pericytes and smooth muscle cells. It has been shown to inhibit adipocyte differentiation, which implicates downregulation of PDGF signaling as a critical event in adipogenesis. Specifically, PDGFRB has been shown to contribute to the remodeling of adipose cells in obese or cold-exposed adult mice.

These Pdgfrb^Flp;fSf-Tomato mice express PDGFRB protein at a level similar to wild type mice and no tdTomato expression is evident prior to Cre recombination. The donating investigator reports that double homozygous mice are viable and fertile.

Cre recombination in these mice leads to the knock-out of Pdgfrb expression and Flp/FRT-mediated intersectional lineage tracing of tdTomato in cre-expressing cells and their progeny. When crossed to Sox2Cre germline cre mice (Stock No. 008454), bR^ΔFlp/+;Sox2-Cre^tg;fSf-Tomato mice have detectable tdTomato expression in white adipocytes and brown adipocytes at P21.

Development

The Pdgfrb^Flp targeting construct (bR-Flp) was designed to replace exons 2-4 of the endogenous platelet derived growth factor receptor, beta (Pdgfrb) gene with (from 5' to 3') a slice acceptor, a loxP-flanked Pdgfrb cDNA, a codon-optimized flpo recombinase sequence (flpo), and an attB/attP-flanked neomycin resistance (neo) cassette. This construct was electroporated into 129S4/SvJaeSor-derived AK7 embryonic stem (ES) cells. Correctly targeted ES cells were transiently transfected with a codon-optimized ΦC3 plasmid to delete the neo cassette. Resulting ES cells, lacking the neo cassette, were injected into blastocysts and the resulting chimeric Pdgfrb^Flp mice were bred to C57BL/6J mice (Stock No. 000664).

On the side, the donating investigator bred Ai65D mice (Stock No. 021875) with Sox2Cre germline Cre mice (Stock No. 008454) to remove the loxP-flanked stop cassette and produce a fluorescent FLP reporter mouse line analogous to Ai65F mice (Stock No. 032864). The Ai65F mice were then bred to Pdgfrb^Flp mice to create the double mutant Pdgfrb^Flp;fSf-Tomato mice. The Pdgfrb^Flp;fSf-Tomato mice were bred to other cre- and cre/ERT2-expressing mouse lines, and these lines were maintained on a mixed genetic background. cre and cre/ERT2 alleles were removed from the double mutant Pdgfrb^Flp;fSf-Tomato mice sent to The Jackson Laboratory as Stock No. 038629. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes.

Allele

Symbol: Gt(ROSA)26Sor^{tm65.2(CAG-tdTomato)Hze}
Name: targeted mutation 65.2, Hongkui Zeng
MGI accession ID: MGI:6260212
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter
Synonyms: Ai65F(RCF-tdT); fSf-Tomato; R26-frt.Stop.frt-tdTomato; Rosa26-tdT-FRT; Ai65F
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Site of expression: Although under control of the endogenous Gt(ROSA)26Sor promoter/enhancer regions and the CAG hybrid promoter, widespread expression of tdTomato is prevented by the STOP cassette. Upon exposure to FLP recombinase, the STOP cassette is removed - resulting in tdTomato fluorescence.
Molecular note: The targeting construct contains (from 5' to 3'): a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a frt-flanked STOP cassette (with a short open reading frame, followed by translational stops in all three reading frames, a synthetic polyA sequence, a polyA sequence from the human growth hormone gene, and a polyA sequence from the Herpes Simplex Virus TK gene), a loxP-flanked STOP cassette (with stop codons in all three reading frames linked to three repeats of the SV40 polyA signal), a sequence encoding the tdTomato fluorescent protein (a non-oligomerizing DsRed fluorescent protein variant with a 12 residue linker fusing two copies of the protein [tandem dimer]), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a bovine growth hormone polyA signal and an AttL site.This entire targeting vector was inserted between exons 1 and 2. PhiC31-mediated recombination removed the AttB/AttP-flanked PGK-Neo-polyA cassette and replace it with the recombined AttB/AttP site (AttL). This entire targeting vector was inserted between exons 1 and 2. PhiC31-mediated recombination removed the AttB/AttP-flanked PGK-Neo-polyA cassette and replaced it with the recombined AttB/AttP site (AttL). Cre-mediated recombination removed the floxed STOP.

Allele

Symbol: Pdgfrb^{tm1.1(flpo)Leol}
Name: targeted mutation 1.1, Lorin E Olson
MGI accession ID: MGI:6728540
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Recombinase-expressing; No functional change
Synonyms: Pdgfrb^Flp
Marker symbol: Pdgfrb
Marker name: platelet derived growth factor receptor, beta polypeptide
Marker synonyms: CD140b; IBGC4; IMF1; JTK12; KOGS; OPDKD; Pdgfr; PDGFR1; PDGFR-1; PENTT
Chromosome: 18
Strain of origin: Not Specified
Molecular note: The targeting cassette consisting of a SA.lox.PDGFRb.lox-Flpo-attB-Neo-attP cassette replaced the exons 2-4 of the endogenous Pdgfrb gene. The PDGFRb is a mouse Pdgfrb gene cDNA. PhiC31 integrase removed the attB-Neo-attP cassette. Cre-mediated recombination is required to achieve expression of the flpo recombinase.