B6N;129S-Prom1^{tm1(cre/ERT2)Gilb}/J

Stock number: 017743
Product name: Prom1C-L
Research areas: Internal/Organ Research, Research Tools, Sensorineural Research

Description

A CreER^T2 fusion protein and β-galactosidase (lacZ) gene knocked into the ATG start codon of Prom1 abolishes gene function in these mice. Useful applications include visualizing and tracking adult stem cell lineages.

Detailed description

A CreER^T2 fusion protein, and an internal ribosome entry site (IRES)-β-galactosidase (lacZ) gene replace the ATG start codon of the prominin 1 (Prom1) gene, abolishing gene expression. PROM1 is a transmembrane glycoprotein expressed on the surface of stem cells. In this strain, endogenous Prom1 promoter/enhancer regions drive lacZ expression in embryonic central nervous system, kidney, intestine, and skeletal system. In adults, expression is seen in acinar and islet cells of the pancreas, goblet and columnar epithelial cells of the colon, photorepectoprs of the eye, renal tube cells, brain, lung, and both male and female reproductive systems. Cre-ER^T2 fusion gene activity is inducible and only observed following tamoxifen administration. When these Prom1^C-L mice are bred with mice containing loxP-flanked sequence, tamoxifen-inducible, Cre-mediated recombination will result in deletion of the floxed sequences in the Prom1-expressing cells of the offspring. For example when bred to Gt(ROSA)26Sor^tm1(EYFP)Cos/J mice (Stock No. 006148), tamoxifen induction results in cre expression in small intestinal crypt cells and the epithelial surface of the villi. Homozygotes are viable and fertile, but are blind due to retinal pigmentosa.

The Cre-ER^T2 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-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

A targeting vector was designed to replace the ATG start codon of the prominin 1 (Prom1) gene with, from 5' to 3', a CreER^T2 fusion protein, an internal ribosome entry site (IRES), nuclear localized β-galactosidase (lacZ), an SV40 polyadenylation signal, and a frt-flanked neomycin (neo) selection cassette. This construct was electroporated into 129S6/SvEv-derived CMTI-1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6 recipient blastocysts and chimeric males were bred with C57BL/6NCrl females. The resulting mice were bred to 129S4/SvJaeSor-Gt(ROSA)26Sor^tm1(FLP1)Dym/J mice (Stock No. 003946) to remove the neo cassette. These Prom1^C-L mice were backcrossed to C57BL/6NCrl for a few generations and then were maintained on a mixed B6;129S background.

Allele

Symbol: Prom1^{tm1(cre/ERT2)Gilb}
Name: targeted mutation 1, Richard J Gilbertson
MGI accession ID: MGI:3830624
Generation method: Targeted
Attributes: Recombinase-expressing; Inducible
Marker symbol: Prom1
Marker name: prominin 1
Chromosome: 5
Strain of origin: 129S6/SvEvTac
Expressed genes: cre/ERT2,Cre recombinase and estrogen receptor 1 (human) fusion gene,
Site of expression: endogenous Prom1 promoter/enhancer regions drive lacZ expression in embryonic central nervous system, kidney, intestine, and skeletal system. In adults, expression is seen in acinar and islet cells of the pancreas, goblet and columnar epithelial cells of the colon, photorepectoprs of the eye, renal tube cells, brain, lung, and both male and female reproductive systems.
Molecular note: The ATG start site of the gene locus was replaced by a cassette containing a creERT2 fusion gene, an IRES-LacZ gene, and a FRT-flanked neomycin selection cassette. Correct targeting was confirmed by PCR. The neomycin selection cassette was later removed by crossing mice with Gt(ROSA)26Sor^tm1(FLP1)Dym mice. Beta-gal expression was detected in the neural tube, rib, kidney, and gut of E10.5-E16.5 heterozygote embryos. Beta-gal expression was also detected in numerous adult tissues including brain, kidney, retina, pancreas, and intestine. Cre expression was also detected in brain, kidney, lung, pancreas, and intestinal epithelium of heterozygote adult mice after tamoxifen administration. Inactivation of the endogenous gene locus was confirmed by RT-PCR of adult homozygous kidney.