B6N.Cg-Tg(Hsd17b1-icre/ERT2)3Casa/J

Stock number: 017310
Research areas: Developmental Biology Research, Research Tools

Description

Mice hemizygous for the BAC Hsd17b1-icreER^T2 transgene are viable, fertile, and normal in size with iCre-ER^T2 expression directed by the hydroxysteroid (17-beta) dehydrogenase 1 (Hsd17b1) promoter/enhancer regions within the BAC transgene. These mice may be useful for studying gene function in ovarian granulose cells.

Detailed description

Mice hemizygous for the BAC Hsd17b1-icreER^T2 transgene are viable, fertile, and normal in size with iCre-ER^T2 expression directed by the hydroxysteroid (17-beta) dehydrogenase 1 (Hsd17b1) promoter/enhancer regions within the BAC transgene. These BAC mice express 8 copies of iCre-ER^T2 protein in granulose cells of the ovarian follicles. iCre-ER^T2 fusion gene activity is inducible and is observed only following tamoxifen administration. When these mice are bred with mice containing loxP-flanked sequence, tamoxifen-inducible, Cre-mediated recombination will result in deletion of the floxed sequences in the BAC-expressing cells of the offspring. These mice may be useful for studying gene function in ovarian granulose cells.

The iCre-ER^T2 fusion protein consists of a codon-improved 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, iCre-ER^T2 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

Bacterial artificial chromosome (BAC) library (CHORI) was used to obtain a 200 kb BAC (#RP24-178B1) containing the entire mouse hydroxysteroid (17-beta) dehydrogenase 1 (Hsd17b1) gene. This BAC was altered to insert an icreER^T2 fusion gene (iCre-ER^T2; a codon improved Cre recombinase fused to a G400V/M543A/L544A triple mutation of the human estrogen receptor ligand binding domain) and a polyadenylation signal (polyA) downstream of the initiation codon of Hsd17b1. This modified BAC was microinjected into fertilized (C57BL/6xCBA) F1 oocytes. Offspring were bred to C57BL/6NTac and founder line 3, containing 8 copies of the Hsd17b1- iCreER^T2 BAC, was established. These mice were subsequently bred with C56BL/6NTac inbred mice for at least 6 generations. Upon arrival at The Jackson Laboratory, mice were bred to C57BL/6NJ inbred mice (Stock No. 005304) for at least one generation.

Allele

Symbol: Tg(Hsd17b1-icre/ERT2)3Casa
Name: transgene insertion 3, Emilio Casanova
MGI accession ID: MGI:4946612
Generation method: Transgenic
Attributes: Recombinase-expressing; Inducible
Marker symbol: Tg(Hsd17b1-icre/ERT2)3Casa
Marker name: transgene insertion 3, Emilio Casanova
Chromosome: UN
Strain of origin: (C57BL/6 x CBA)F1
Expressed genes: cre,cre recombinase,bacteriophage P1; ESR1,estrogen receptor 1,human
Site of expression: When mice carrying this transgene are bred with mice containing loxP-flanked sequence, tamoxifen-inducible, Cre-mediated recombination will result in deletion of the floxed sequences in granulose cells of the ovarian follicles.
Molecular note: A 200kb BAC containing the Hsd17b1 gene was modified by homologous recombination to generate the construct. The open reading frame sequence for codon-improved cre recombinase (icre) fused to a mutated ligand-binding domain (LBD) of the human estrogen receptor (ERT2) was inserted into the exon containing the transcription initiation codon of the Hsd17b1 gene. The initial targeting vector also contained the bovine growth hormone polyA signal and an Frt-flanked ampicillin cassette. The amp marker was subsequently removed from targeted by clones upon electroporation of a plasmid expressing Flp recombinase. Purified BAC clones were injected into (C57BL/6 x CBA)F1 oocytes. Three lines expressing the transgene were generated. Lines 1 and 2 were shown to contain 2 copies of the insert by Southern blot, while line 3 had 8 copies. Line 3 mRNA levels of icre/ERT2 were highest in ovaries and this line was used for subsequent analyses.