B6.129S(Cg)-Id2^{tm1.1(cre/ERT2)Blh}/ZhuJ

Stock number: 016222
Product name: Id2-CreER^T2
Research areas: Cell Biology Research, Developmental Biology Research, Endocrine Deficiency Research, Hematological Research, Immunology, Inflammation and Autoimmunity Research, Internal/Organ Research, Reproductive Biology Research, Research Tools

Description

This Id2-CreERT2 knock-in allele was designed to both abolish Id2 gene function and direct CreER^T2 fusion protein expression from the endogenous Id2 promoter/enhancer elements. Cre-ERT2 fusion gene activity is inducible by tamoxifen administration.

Detailed description

The Id2-CreERT2 knockin allele was designed to both abolish inhibitor of DNA binding 2 (Id2) gene function and express CreER^T2 fusion protein from the Id2 promoter/enhancer elements. Cre-ERT2 fusion gene activity is inducible and can be observed following tamoxifen administration. As such, when Id2-CreERT2 knockin mice are bred with mice containing loxP-flanked sequences, tamoxifen-inducible Cre-mediated recombination will result in deletion of the floxed sequences in the Id2-expressing cells of the offspring.

No mRNA or protein expression from the Id2-CreERT2 allele is observed. The donating investigator reports that homozygous mice are runted with defective lung alveolarization. Other organ systems have not been evaluated. However, Id2-CreERT2 homozygotes may be expected to exhibit the same phenotype as mice homozygous for other null mutations of this gene (including postnatal lethality and defects of the immune system, digestive tract, kidneys, adipose tissue and mammary gland development).Heterozygous mice are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities. The donating investigator also reports tamoxifen-inducible Cre recombinase activity recapitulates the endogenous Id2 expression pattern. Following tamoxifen administration, Cre recombinase activity recapitulates the epithelial expression of the endogenous gene (distal tip lung epithelial multipotent precursor cells of the developing lung throughout branching morphogenesis up to embryonic day (E)16.5). Additionally, the Cre recombinase activity in immune cells (during lymphopoiesis, hematopoiesis, or adaptive immune responses) was not directly assessed by the donating investigator. Cre recombinase activity is not observed in lung tissue prior to tamoxifen treatment(other systems not examined).

Of note, the same group designed an identically-targeted Id2-eGFP knockin mouse line to express an enhanced green fluorescent protein (rather than Cre-ERT2 fusion protein) from the Id2 promoter/enhancer elements. That Id2-eGFP knockin mouse line is available as Stock No. 016224.

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.

Development

The Id2-CreERT2 knockin mutation was created by the laboratory of Dr. Brigid LM Hogan (Duke University Medical Center). A targeting vector was designed to insert a CreER^T2 fusion gene (followed by an frt-flanked PGK-neo cassette) into the translation initiation codon of the inhibitor of DNA binding 2 locus (Id2); replacing most of exon 1 except the final 25 nucleotides. The CreER^T2 fusion gene (Cre-ER^T2) is Cre recombinase fused to a G400V/M543A/L544A triple mutation of the human estrogen receptor ligand binding domain. The targeting construct was electroporated into 129SvEv-derived embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts and chimeric mice were bred with C57BL/6NCrl mice to originate the colony. Mutant mice were bred with ROSA26-FLPe mice (on an uncharacterized genetic background) to remove the PGK-neo selection cassette. These Id2-CreERT2 knockin mice were subsequently bred to C57BL/6NCrl mice for 5-8 generations (and the ROSA26-FLPe was removed). Next, heterozygous mutant mice were sent to Dr. Yuan Zhang (Duke University) where they were backcrossed to C57BL/6J mice (see SNP note below) for at least one additional generation prior to sending to The Jackson Laboratory Repository. Upon arrival, Id2-CreERT2 knockin mice were bred with C57BL/6NJ inbred mice (Stock No. 005304) for at least one generation to establish the Id2-CreERT2 knockin colony. During backcrossing, the donating investigator reports that the Y chromosome was fixed to the C57BL/6J genetic background.

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, 4 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.

Allele

Symbol: Id2^{tm1.1(cre/ERT2)Blh}
Name: targeted mutation 1.1, Brigid L Hogan
MGI accession ID: MGI:4398923
Generation method: Targeted
Attributes: Recombinase-expressing; Inducible
Synonyms: Id2^{tm1(CRE/ESR1)Blh}; Id2-CreER^T2
Marker symbol: Id2
Marker name: inhibitor of DNA binding 2
Chromosome: 12
Strain of origin: 129S/SvEv
Expressed genes: cre/ERT2,Cre recombinase and estrogen receptor 1 (human) fusion gene,
Site of expression: Following tamoxifen administration, Cre recombinase activity recapitulates the epithelial expression of the endogenous gene (distal tip lung epithelial multipotent precursor cells of the developing lung throughout branching morphogenesis up to embryonic day (E)16.5).
Molecular note: The targeting vector contained an cre/ERT2 cassette inserted at the translation initiation codon of the Id2 locus, replacing most of exon 1, except for the final 25 nucleotides. The cre cassette was followed by a frt-PGK-neo-frt cassette, which was removed by flp-mediated excision after germline transmission.