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

Stock number: 038627
Product name: Pdgfra^K.Flp;fSf-Tomato
Strain synonyms: aR-K.Flp;fSf-Tomato
Research areas: Research Tools

Description

Pdgfra^K.Flp;fSf-Tomato knock-in/knock-out mice have a floxed stop cassette and cDNA encoding a constitutively active Pdgfra gene (D842V; PDGFRα^K) with a T2A-flpo sequence replacing the PDGFRα^K stop codon. Mice also contain a conditional tdTomato fluorescent FLP reporter allele. Cre recombination leads to the activation of PDGFRA kinase activity and Flp/FRT-mediated lineage tracing in cre-expressing cells.

Detailed description

Pdgfra^K.Flp;fSf-Tomato knock-in/knock-out mice have a floxed stop cassette and cDNA encoding a constitutively active platelet derived growth factor receptor, alpha (Pdgfra) gene with a D842V mutation in the kinase domain (PDGFRα^K) and a T2A-flpo sequence replacing the PDGFRα^K stop codon. Mice also contain a conditional tdTomato fluorescent FLP reporter allele, Ai65F or fSf-Tomato. Pdgfra is expressed in a wide array of mesenchymal tissues, including the lung, heart, intestine, skin, and cranial facial mesenchyme. It has been shown to inhibit adipocyte differentiation, which implicates downregulation of PDGF signaling as a critical event in adipogenesis. Aberrant PDGFRA signaling inhibits adipogenesis and promotes fibrosis, and has been linked to a large number of adult diseases including fibrosis and cancer, and pediatric diseases such as bronchopulmonary dysplasia (BPD).

These Pdgfra^K.Flp;fSf-Tomato mice express PDGFRA protein at a level slightly reduced compared to wildtype mice and no tdTomato expression is evident prior to Cre recombination. The donating investigator reports that mice heterozygous for Pdgfra^K.Flp and homozygous for fSf-Tomato are viable and fertile. Mice homozygous for the Pdgfra^K.Flp allele are functionally null and embryonic lethal in the absence of Cre.

Cre recombination in these mice leads to the expression of a constitutively active PDGFRA protein and Flp/FRT-mediates intersectional lineage tracing of tdTomato in cre-expressing cells and their progeny. When crossed to Sox2Cre germline Cre mice (Stock No. 008454), aR^ΔK.Flp/+;Sox2-Cre^tg;fSf-Tomato embryos die after E13.5 with broad expression of tdTomato in mesenchymal cells of the frontonasal prominence but not in cells of the nasal epithelium, the olfactory lobe, or the overlying epidermis.

Development

The Pdgfra^K.Flp targeting construct (aR-K.Flp) was designed to replace exons 2-4 of the endogenous platelet derived growth factor receptor, alpha (Pdgfra) gene with (from 5' to 3') a splice acceptor, a loxP-flanked neomycin resistance (neo) cassette and STOP cassette, and Pdgfra cDNA that contains an aspartic acid to valine gain-of-function mutation at codon 842 (D842V) mutation (PDGFRα^K), and a T2A self-cleaving peptide followed by a codon-optimized flpo recombinase sequence (flpo) replacing the PDGFRα^K stop codon. This construct was electroporated into 129S4/SvJaeSor-derived AK7 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts and the resulting chimeric Pdgfra^K.Flp mice were 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 Pdgfra^K.Flp mice to create the double mutant Pdgfra^K.Flp;fSf-Tomato mice. The Pdgfra^K.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 Pdgfra^K.Flp;fSf-Tomato mice sent to The Jackson Laboratory as Stock No. 038627. 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: Pdgfra^{tm1(flpo)Leol}
Name: targeted mutation 1, Lorin E Olson
MGI accession ID: MGI:6728538
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Recombinase-expressing; Null/Knockout
Synonyms: Pdgfra^K.Flp
Marker symbol: Pdgfra
Marker name: platelet derived growth factor receptor, alpha polypeptide
Marker synonyms: APDGFR; CD140a; PDGFACE; PDGFR2; PDGFR-2
Chromosome: 5
Strain of origin: Not Specified
Molecular note: The targeting cassette consisting of a SA.lox.Neo/Stop.lox-PDGFRaK.T2A.Flp cassette replaced the exons 2-4 of the endogenous Pdgfra gene. The PDGFRaK is a mouse Pdgfra gene cDNA with a point mutation (D842V) renders it constitutively active. Cre-mediated recombination is required to achieve expression of the constitutively active Pdgfra gene with a point mutation (D842V) and the flpo recombinase. In cre naive state it is a null allele.