B6.Cg-Pip^{tm1.1(cre/ERT2)Ovi}/J

Stock number: 023201
Research areas: Endocrine Deficiency Research, Research Tools

Description

Pip^GCE mice express an EGFP/Cre-ER^T2 fusion protein in acinar cells of the neonatal and adult submandibular gland. These mice may have applications in studies requiring conditional gene manipulation in the submandibular gland, as well as for acinar cell lineage fate mapping.

Detailed description

The prolactin-induced protein encoded by the Pip gene is secreted by apocrine cells into exocrine fluids. Prolactin-induced protein binds actin and is a biomarker for breast cancer. These knock-in mice express the GCE cassette (an EGFP/creERT2 fusion gene) from the endogenous Pip locus. When Pip^GCE mice are bred with mice containing loxP-flanked sequence, tamoxifen-inducible, cre-mediated recombination will result in deletion of the floxed sequences. Tamoxifen administration in double mutant mice, carrying this transgene and a reporter, induces Cre recombination in acinar cells of the submandibular gland. Although Pip is expressed as early as E14 in proacinar cells, cre activity is not detected in E15.5 or E17.5 submandibular gland. Tamoxifen inducible cre recombinase activity is not detected in parotid, sublingual or lacrimal glands, kidney, lung, pancreas, prostate, or ovary tissues. The Donating Investigator has not made the strain homozygous, although homozygotes are predicted to be viable and fertile. EGFP expression is not detected by direct fluorescence or anti-GFP antibody (immunofluorescence). During backcrossing, the Y chromosome may not have been fixed to the C57BL/6J genetic background.

The Cre-ERT2 fusion protein consists of Cre recombinase fused to a triple mutant form of the human estrogen receptor, which does not bind its natural ligand (17β-estradiol) at physiological concentrations, but will bind the synthetic estrogen receptor ligands 4-hydroxytamoxifen (OHT or tamoxifen) and, with lesser sensitivity, ICI 182780. Restricted to the cytoplasm, Cre-ERT2 can only gain access to the nuclear compartment after exposure to tamoxifen. To counteract the mixed estrogen agonist effects of tamoxifen injections, which can result in late fetal abortions in pregnant mice, progesterone may be coadministered.

Development

A targeting construct containing the GCE cassette (EGFP and cre/ERT2 fusion gene) followed by SV40 polyadenylation sequence and an FRT-flanked neo cassette was used to disrupt exon 1 of the targeted gene. The construct was electroporated into (129S6/SvEvTac x C57BL/6NCrl)F1-derived G4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6J blastocysts, and chimeras were bred to C57BL/6J mice. The resulting Pip-GCE-Neo heterozygotes were then bred to Actin-Flippase mouse strain on a B6.129S4 genetic background (Stock No. 009086) to remove the FRT-flanked NEO cassette, generating Pip^GCE offspring. The mice were then backcrossed to C57BL/6 for 4 generations. During backcrossing, the Y chromosome may not have been fixed to the C57BL/6J genetic background. Upon arrival, B6N4 sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Pip^{tm1.1(cre/ERT2)Ovi}
Name: targeted mutation 1.1, Catherine E Ovitt
MGI accession ID: MGI:5661584
Generation method: Targeted
Attributes: Recombinase-expressing; Inducible
Marker symbol: Pip
Marker name: prolactin induced protein
Chromosome: 6
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Expressed genes: cre/ERT2,Cre recombinase and estrogen receptor 1 (human) fusion gene,
Site of expression: Tamoxifen administration results in cre expression from an EGFP/creERT2 fusion gene in acinar cells of the submandibular gland.
Molecular note: The eGFP-CreERT2 (GCE) fragment with an SV40 poly adenylation site and neomycin resistance cassette were inserted immediately downstream of the translational initiation codon ATG. The knock-in construct removed the coding sequences from exon 1 and placed the GCE gene under the control of the endogenous regulatory sequences. Flp-mediated recombination removed the selection cassette. The EGFP could not be detected by direct fluorescence or anti-GFP antibody staining.