C57BL/6-Spp2^{tm1(cre/ERT2)Amc}/J

Stock number: 025209
Research areas: Developmental Biology Research, Endocrine Deficiency Research, Internal/Organ Research, Research Tools

Description

The Spp2^G2aCE knock-in allele was designed to both abolish Spp2 gene expression and express EGFP and Cre-ER^T2 proteins from the Spp2 promoter in proximal tubule cells of the mouse kidney.

Detailed description

The Spp2^G2aCE knock-in allele was designed to both abolish secreted phosphoprotein 2 (Spp2) gene expression and express individual EGFP and Cre-ER^T2 proteins from the Spp2 promoter/enhancer elements. SPP2 encodes a serum phosphoprotein synthesized by the liver which is involved in bone growth, repair, and turnover. Heterozygous Spp2^G2aCE mice are viable and fertile. The donating investigator has not attempted to make this strain homozygous, and mice may still be segregating for the A^tm1Brd allele. Weak EGFP fluorescence is seen in proximal tubule cells of the mouse kidney and in liver cells at 19.5 days post coitum. Cre-ER^T2 gene activity is inducible and can be observed following tamoxifen administration. As such, when these mice are bred with mice containing loxP-flanked sequences, tamoxifen-inducible Cre-mediated recombination will result in deletion of the floxed sequences in proximal tubule cells in the embryonic and adult kidney.

This strain was transferred from the collection of the GenitoUrinary Development Molecular Anatomy Project (GUDMAP).

Development

An exchange vector was designed to be inserted downstream of exon 1 of the secreted phosphoprotein 2 (Spp2) gene using dual-recombinase mediated cassette exchange (dRMCE). Specifically, the exchange vector contained (from 5’ to 3’) a frt site, a viral F2A oligopeptide (to mediate ribosomal skipping), an enhanced green fluorescent protein (EGFP) sequence, a viral T2A oligopeptide, and a Cre-ER^T2 sequence (Cre recombinase fused to a G525R mutant form of the mouse estrogen receptor ligand binding domain). A rox-flanked puromycin resistance (puro) cassette and a loxP site were inserted at the 3' end of the exchange vector.

This exchange vector (along with the pDIRE plasmid containing an icre-expression cassette and a Flpo-expression cassette to facilitate insertion of the exchange vector) was electroporated into C57BL/6N-A^tm1Brd-derived JM8A3.N1 knockout first reporter embryonic stem (ES) cells obtained from the European Conditional Mouse Mutagenesis (EUCOMM) consortium containing clone EPDO558_1_A04. Recombined ES cells, with the neo removed and resistant to puro, were injected into B6(Cg)-Tyr^c-2J/J blastocysts and resulting chimeric mice were bred to C57BL/6J to establish a colony. Upon arrival at The Jackson Laboratory, mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation to establish the colony. Mice may still be segregating for the A^tm1Brd allele.

Allele

Symbol: Spp2^{tm1(cre/ERT2)Amc}
Name: targeted mutation 1, Andrew P McMahon
MGI accession ID: MGI:5565344
Generation method: Targeted
Attributes: Recombinase-expressing; Reporter; Inducible; Null/Knockout; RMCE-ready
Marker symbol: Spp2
Marker name: secreted phosphoprotein 2
Chromosome: 1
Strain of origin: C57BL/6N-A^tm1Brd
Expressed genes: GFP,Green Fluorescent Protein,; cre/ERT2,Cre recombinase and estrogen receptor 1 (human) fusion gene,
Site of expression: When mice carrying this allele are bred with mice containing loxP-flanked sequences, tamoxifen-inducible Cre-mediated recombination will result in deletion of the floxed sequences in proximal tubule cells in the embryonic and adult kidney.
Molecular note: The allele was generated by a dual recombinase-mediated cassette exchange protocol. The exchange vector contained (from 5' to 3') an frt site, a viral F2A oligopeptide (to mediate ribosomal skipping), an enhanced green fluorescent protein (EGFP) sequence, a viral T2A oligopeptide, and a cre/ERT2 sequence. A rox-flanked puromycin resistance (puro) cassette and a loxP site were inserted at the 3' end of the exchange vector.
General note: This exchange vector (along with the pDIRE plasmid containing an icre-expression cassette and a Flpo-expression cassette to facilitate insertion of the exchange vector) was electroporated into C57BL/6N-Atm1Brd-derived JM8A3.N1 knockout first reporter embryonic stem (ES) cells obtained from the European Conditional Mouse Mutagenesis (EUCOMM) consortium containing clone EPDO558_1_A04). Recombined ES cells, with the neo removed and resistant to puro, were injected into B6(Cg)-Tyrc-2J/J blastocysts and resulting chimeric mice were bred to C57BL/6J to establish a colony. Recombined ES cells, with the neo removed and resistant to puro, were injected into B6(Cg)-Tyrc-2J/J blastocysts and resulting chimeric mice were bred to C57BL/6J to establish a colony.