B6;129S-Tfcp2l1^{tm1.1(cre/ERT2)Ovi}/J

Stock number: 028732
Product name: Tcfcp2l1^GCE
Research areas: Endocrine Deficiency Research, Research Tools

Description

Tcfcp2l1^GCE knock-in mice express a EGFP/cre/ERT2 fusion protein in acinar and duct cells of the salivary gland, and in the lacrimal glands. These mice may have applications in studies requiring conditional gene manipulation in the salivary gland, as well as in acinar or duct cell lineage fate mapping.

Detailed description

Transcription factor CP2-like 1, encoded by the Tcfcp2l1 gene, is required for differentiation of duct cells in the salivary gland and kidney, and is important in self-renewal of embryonic stem cells. These knock-in mice express the GCE cassette (an EGFP/creERT2 fusion gene) from the endogenous Tcfcp2l1 locus. When Tcfcp2l1^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 GCE cassette and a reporter, induces Cre recombination in duct cells of the salivary gland, lacrimal glands, kidney cortex and in adult lung bronchial cells. Expression of the reporter was also induced in acinar cells. No inducible cre recombinase activity was detected in embryonic salivary glands. EGFP expression is not detected by direct fluorescence or anti-GFP antibody (immunofluorescence). Heterozygotes are viable and fertile. The Donating Investigator has not made the strain homozygous. During backcrossing, the Y chromosome may not have been fixed to the C57BL/6J genetic background.

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 GCE cassette was inserted at the initiation site in exon 1 of the Tcfcp2l1 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 Tcfcp2l1-GCE-Neo allele 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 Tcfcp2l1^GCE offspring. The mice were then backcrossed to C57BL/6 for 1 generation to breed out the FLP recombinase allele. 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).

A 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. Numerous markers throughout the genome were segregating, suggesting an incomplete backcross.

Allele

Symbol: Tfcp2l1^{tm1.1(cre/ERT2)Ovi}
Name: targeted mutation 1.1, Catherine E Ovitt
MGI accession ID: MGI:5662395
Generation method: Targeted
Attributes: Recombinase-expressing; Reporter; Inducible
Marker symbol: Tfcp2l1
Marker name: transcription factor CP2-like 1
Chromosome: 1
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 duct cells of the salivary gland, lacrimal glands, kidney cortex and in adult lung bronchial cells.
Molecular note: The targeting construct was generated by inserting the diphtheria toxin A gene (DTA, for positive selection), 3.2 kb 5 homologous arm containing the 5' UTR of exon 1, and 5.5 kb 3 homologous arm containing exon 2 into pBluescript SKII(+). Then the eGFP-CreERT2 (GCE) fragment (McMahon et al. 2008) 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 endogenous regulatory sequences. Flp-mediated recombination removed the selection cassette. The EGFP could not be detected by direct fluorescence or anti-GFP antibody staining.