B6.Cg-Ascl3^{tm2.1(icre/ESR1*)Ovi}/J

Stock number: 038129
Product name: Ascl3^P2A-GCE
Research areas: Cell Biology Research, Research Tools, Sensorineural Research

Description

Ascl3^P2A-GCE knock-in mice express an iCreERT fusion protein in achaete-scute family bHLH transcription factor 3 (Ascl3)-expressing cells while retaining endogenous Ascl3 activity. When these mice are bred with mice containing a loxP-flanked sequence of interest, tamoxifen-inducible, Cre recombinase activity is detected in the Ascl3-expressing cells of the offspring. Of note, the donating investigator has indicated that eGFP was included in the target construct but expression is not detectable either by direct fluorescence or immunohistochemistry.

Detailed description

The achaete-scute family of bHLH transcription factors are involved in cell fate determination and differentiation. Achaete-scute family transcription factor 3 (encoded by Ascl3) is expressed in progenitor cells of embryonic salivary glands and in a subset of salivary gland duct cells postnatally and in adult mice. Ascl3 expression is part of a conserved gene signature for a novel sensory cell type known as ionocyte in pulmonary epithelium. Ionocytes may be a new therapeutic target for cystic fibrosis and Sjögrens syndrome.

Ascl3^P2A-GCE knock-in mice express a P2A ribosomal skip cleavage peptide sequence followed by an eGFP/tamoxifen-inducible codon-optimized Cre recombinase fusion protein under the control of the endogenous Ascl3 protomer. When Ascl3^P2A-GCE knock-in mice are bred with mice containing loxP-flanked sequences, tamoxifen-inducible Cre-recombinase activity is detected in Ascl3-expressing cells of the offspring. Unlike B6.129S1(Cg)-Ascl3^{tm1.1(EGFP/cre)Ovi}/J (Stock No. 021794) mice, this Ascl3^P2A-GCE knock-in line is believed to retain endogenous Ascl3 expression.

Heterozygous Ascl3^P2A-GCE knock-in mice are viable and fertile with no overt phenotype. The donating laboratory has not evaluated the viability of homozygous mice to date. The donating investigator has indicated that eGFP expression is not detectable either by direct fluorescence or immunohistochemistry. After crossing these Ascl3^P2A-GCE knock-in mice to B6.Cg-Gt(ROSA)26Sor^{tm9(CAG-tdTomato)Hze}/J (Stock No. 007909), RFP was detected as expected in Ascl3 expressing cells of the submandibular, parotid and sublingual glands, as well as in lacrimal glands and embryonic E17.5 lung tissue. The donating laboratory has also verified Cre-recombinase activity by crossing Ascl3^P2A-GCE knock-in mice to B6.129S6(Cg)-Gt(ROSA)26Sor^tm1(DTA)Jpmb/J (Stock No. 032087), which resulted in successful ablation of Ascl3-expressing ionocytes after tamoxifen induction. Endogenous Ascl3 mRNA expression is retained prior to tamoxifen induction. These knock-in mice may be useful for studying the expression and function of Ascl3-expressing cells.

Note, if additional characterization of these mice is performed, we may modify the strain description accordingly. Based on sequencing analysis performed by The Jackson Laboratory (March 2024) no GFP sequence was found. Additionally, the tamoxifen-inducible cre sequence was found to have human ER sequence.

Development

The Ascl3^P2A-GCE knock-in targeting vector consisted of a P2A sequence followed by an eGFP/iCreERT fusion protein and an FRT-flanked PGK-neomycin selection cassette. The targeting construct was inserted immediately upstream of the termination codon of the achaete-scute family bHLH transcription factor 3 (Ascl3) gene. The construct was electroporated into (129S6/SvEvTac x C57BL/6NCr) F1-derived G4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts and the resulting chimeric mice were identified by genotyping and bred to B6.129S4-Gt(ROSA)26Sor^tm1(FLP1)Dym/RainJ (Stock No. 009086) to remove the neo cassette. The FLP1 transgene was removed by further breeding to C57BL/6J mice (Stock No. 000664). The resulting colony of Ascl3^P2-GCE heterozygous mice was maintained on a C57BL/6J background for at least 6 generations by the donating laboratory. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes.

Of note, based on sequencing analysis performed by The Jackson Laboratory (March 2024) no GFP sequence was found. Additionally, the tamoxifen-inducible cre sequence was found to have human ER sequence.

Allele

Symbol: Ascl3^{tm2.1(icre/ESR1*)Ovi}
Name: targeted mutation 2.1, Catherine E Ovitt
MGI accession ID: MGI:7447443
Generation method: Targeted
Attributes: Recombinase-expressing; Reporter; Inducible
Marker symbol: Ascl3
Marker name: achaete-scute family bHLH transcription factor 3
Marker synonyms: bHLHa42; HASH3; ICRFP703B1614Q5.4; ICRFP703N2430Q5.4; Sgn1
Chromosome: 7
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Molecular note: The targeting vector consists of a P2A sequence followed by an icre (codon-optimized cre) /mutated human ESR1 fusion protein and an FRT-flanked PGK-neomycin selection cassette. The targeting construct was inserted immediately upstream of the termination codon of the achaete-scute family bHLH transcription factor 3 (Ascl3) gene. The neo-selection cassette was removed by Flp recombinase.