JAX
Mouse Strain Datasheet - 008875
B6.129P2-Lgr5tm1(cre/ERT2)Cle/J
Also Known As: Lgr5-EGFP-IRES-creERT2
Heterozygous mice harbor an Lgr5-EGFP-IRES-creERT2 "knock-in" allele that both abolishes Lgr5 gene function and expresses EGFP and CreERT2 fusion protein. When these mice mice are bred with mice containing a loxP-flanked sequence of interest, tamoxifen-inducible, Cre-mediated recombination will result in deletion of the floxed sequences in the Lgr5-expressing cells of the offspring. These mice may be useful for lineage-tracing or marking Lgr5-expressing stem cells of the small intestine.
Donating Investigator
Hans Clevers, Hubrecht Institute
Genetic overview
| Genetic Background | Generation |
|---|---|
|
N11+N3F5
|
Lgr5tm1(cre/ERT2)Cle
| Allele Type | Gene Symbol | Gene Name |
|---|---|---|
| Targeted (Inducible, Recombinase-expressing, Reporter) | Lgr5 | leucine rich repeat containing G protein coupled receptor 5 |
Research Applications
- Cancer Research
- Internal/Organ Research
- Research Tools
Base Price
Starting at:
| $255.00 Domestic price for female |
| $329.00 Domestic price for breeder pair |
Important Note
The donating investigator reports variegated expression of the Lgr5-EGFP-IRES-CreERT2 transgene in the small intestine and colon (something which may happen often with genes that are expressed early during intestinal development). This variegated expression is advantageous for performing clonal lineage tracing and sorting intestinal stem cells, but may have limitations for more quantitative studies such as Lgr5-Cre driven knockout strategies.
Detailed Description
While homozygous mice are not viable, heterozygous Lgr5-EGFP-IRES-CreERT2 mice are viable and fertile; harboring a Lgr5-EGFP-IRES-creERT2 "knock-in" allele that both abolishes Lgr5 (Gpr49) gene function and expresses EGFP and CreERT2 fusion protein from the Lgr5 promoter/enhancer elements. EGFP fluorescence is observed in crypt base columnar cells in small intestine (aka stem cells of the small intestine) and colon. Cre-ERT2 fusion gene activity is inducible; observed in the same cells only following tamoxifen administration. EGFP or inducible CreERT2 expression may also be observed in other Lgr5-expressing cell types (including pre-malignant mouse adenomas, colon cancer cells, epithelial stem cells of the stomach gland, basal epithelial layer stem cells of the mammary glands, and hair follicle stem cells).
The donating investigator reports variegated expression of the Lgr5-EGFP-IRES-CreERT2 transgene in the small intestine and colon (something which may happen often with genes that are expressed early during intestinal development). This variegated expression is advantageous for performing clonal lineage tracing and sorting intestinal stem cells, but may have limitations for more quantitative studies such as Lgr5-Cre driven knockout strategies.
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. As such, when Lgr5-EGFP-IRES-creERT2 mice are bred with mice containing loxP-flanked sequence of interest, tamoxifen-inducible, Cre-mediated recombination will result in deletion of the floxed sequences in the Lgr5-expressing cells of the offspring.
View cre expression characterization.
Development
The EGFP-IRES-creERT2 targeting construct was designed to insert an enhanced green fluorescent protein sequence (EGFP), an internal ribosome entry site (IRES), a CreERT2 fusion gene (Cre-ERT2; Cre recombinase fused to a G400V/M543A/L544A triple mutation of the human estrogen receptor ligand binding domain), a polyA signal, and a loxP-flanked neo cassette into the first ATG codon of the targeted gene. This construct was inserted into the targeted gene via electroporation into male 129P2/OlaHsd-derived IB10/E14IB10 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts and chimeric males were bred with C57BL/6 females. Mutant mice were then crossed to EIIa-cre mice (C57BL/6 genetic background; see Stock No. 003724) to remove the neo selection cassette. The donating investigator reported that the resulting Lgr5-EGFP-IRES-creERT2 mice (floxed-neo removed) were then backcrossed to C57BL/6J (see SNP note below) for at least 4 generations. Next, Lgr5-EGFP-IRES-creERT2 mice were bred with Rosa26-lacZ mice (C57BL/6 genetic background; see Stock No. 003474). Mice with both mutations were further backcrossed for more than 6 generations. Upon arrival at The Jackson Laboratory, the mice were bred with C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the colony. Mice were then selectively bred to remove the Rosa26-lacZ allele.
A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 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. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, 2 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J ; C57BL/6N genetic background.
Expression Data
| Expressed Gene | GFP, Green Fluorescent Protein, |
|---|---|
| Expressed Gene | cre/ERT2, Cre recombinase and estrogen receptor 1 (human) fusion gene, |
| Site of Expression | Variegated EGFP fluorescence is observed in crypt base columnar cells in small intestine (stem cells of the small intestine) and colon. |
Control Suggestions
- Wild-type from the colony
- 000664 C57BL/6J
Additional Information
Selected References
- Barker N; van Es JH; Kuipers J; Kujala P; van den Born M; Cozijnsen M; Haegebarth A; Korving J; Begthel H; Peters PJ; Clevers H. 2007. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449(7165):1003-7. PubMed: 17934449MGI: J:127123
Lgr5tm1(cre/ERT2)Cle
Allele Symbol: Lgr5tm1(cre/ERT2)Cle
| Allele Name | targeted mutation 1, Hans Clevers |
|---|---|
| Allele Type | Targeted (Inducible, Recombinase-expressing, Reporter) |
| Allele Synonym(s) | Lgr5 EGFP-IRES-creERT2; Lgr5-EGFP; Lgr5-EGFP-IRES-creERT2; Lgr5-cre; Lgr5-creERT; Lgr5tm1(cre/ESR1)Cle |
| Gene Symbol and Name | Lgr5, leucine rich repeat containing G protein coupled receptor 5 |
| Gene Synonym(s) | FEX; G protein-coupled receptor 49; GPR49; GPR67; GRP49; Gpr49; Gpr49; HG38 |
| Expressed Gene | GFP, Green Fluorescent Protein, |
| Expressed Gene | cre/ERT2, Cre recombinase and estrogen receptor 1 (human) fusion gene, |
| Site of Expression | Variegated EGFP fluorescence is observed in crypt base columnar cells in small intestine (stem cells of the small intestine) and colon. |
| Strain of Origin | 129P2/OlaHsd |
| Chromosome | 10 |
| Molecular Note | An EGFP-IRES-creERT2 cassette was inserted into exon 1 of the gene along with a loxP-flanked neomycin cassette. The neomycin gene was then excised in vivo by crossing with cre-deleter strain. GFP expression is driven by the endogenous promoter. Cre expression is induced in Lgr5 expressing cells by tamoxifen treatment. |
| Mutations Made By | Nick Barker, Hubrecht Institute |
Disease Terms
Research Areas By Genotype
This mouse can be used to support research in many areas including:
- Cancer Research
- Other
- tumor metastasis
- Other
- Internal/Organ Research
- Gastrointestinal Defects
- Research Tools
- Cancer Research
- Cre-lox System
- Cre Recombinase Expression
- Cre Recombinase Expression: Inducible
- Developmental Biology Research
- Cre-lox System
- transplantation marker for embryonic and adult tissue
- Fluorescent Proteins
- Genetics Research
- Mutagenesis and Transgenesis
- Mutagenesis and Transgenesis: Cre-lox System
- Tissue/Cell Markers
- Tissue/Cell Markers: Cre-lox System
- Tissue/Cell Markers: multiple
- Tissue/Cell Markers: transplantation marker for embryonic and adult tissue
- Toxicology Research
- xenograft/transplant host
Genotype: GFP related
- Research Tools
- Fluorescent Proteins
References
- Barker N; van Es JH; Kuipers J; Kujala P; van den Born M; Cozijnsen M; Haegebarth A; Korving J; Begthel H; Peters PJ; Clevers H. 2007. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449(7165):1003-7. PubMed: 17934449MGI: J:127123
Additional - Lgr5tm1(cre/ERT2)Cle related
- Afrazi A; Branca MF; Sodhi CP; Good M; Yamaguchi Y; Egan CE; Lu P; Jia H; Shaffiey S; Lin J; Ma C; Vincent G; Prindle T Jr; Weyandt S; Neal MD; Ozolek JA; Wiersch J; Tschurtschenthaler M; Shiota C; Gittes GK; Billiar TR; Mollen K; Kaser A; Blumberg R; Hackam DJ. 2014. Toll-like receptor 4-mediated endoplasmic reticulum stress in intestinal crypts induces necrotizing enterocolitis. J Biol Chem 289(14):9584-99. PubMed: 24519940MGI: J:212438
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- Barker N; Ridgway RA; van Es JH; van de Wetering M; Begthel H; van den Born M; Danenberg E; Clarke AR; Sansom OJ; Clevers H. 2009. Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 457(7229):608-11. PubMed: 19092804MGI: J:144216
- Barry ER; Morikawa T; Butler BL; Shrestha K; de la Rosa R; Yan KS; Fuchs CS; Magness ST; Smits R; Ogino S; Kuo CJ; Camargo FD. 2013. Restriction of intestinal stem cell expansion and the regenerative response by YAP. Nature 493(7430):106-10. PubMed: 23178811MGI: J:194212
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- Callejas-Valera JL; Iglesias-Bartolome R; Amornphimoltham P; Palacios-Garcia J; Martin D; Califano JA; Molinolo AA; Gutkind JS. 2016. mTOR inhibition prevents rapid-onset of carcinogen-induced malignancies in a novel inducible HPV-16 E6/E7 mouse model. Carcinogenesis 37(10):1014-25. PubMed: 27538837MGI: J:235431
- Cammareri P; Rose AM; Vincent DF; Wang J; Nagano A; Libertini S; Ridgway RA; Athineos D; Coates PJ; McHugh A; Pourreyron C; Dayal JH; Larsson J; Weidlich S; Spender LC; Sapkota GP; Purdie KJ; Proby CM; Harwood CA; Leigh IM; Clevers H; Barker N; Karlsson S; Pritchard C; Marais R; Chelala C; South AP; Sansom OJ; Inman GJ. 2016. Inactivation of TGFbeta receptors in stem cells drives cutaneous squamous cell carcinoma. Nat Commun 7:12493. PubMed: 27558455MGI: J:241617
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