B6;129X1-Gt(ROSA)26Sor^tm2(ATF4)Myz/J

Stock number: 029394
Product name: Rosa26-ATF4^{loxtg
Research areas: Research Tools}

Description

Rosa26-ATF4^loxtg mutant mice carry a floxed stop/FLAG-tagged human ATF4 cassette knocked into the mouse Gt(ROSA)26Sor gene. Cre-mediate excision of the floxed stop enables site-directed expression of FLAG-tagged human ATF4 protein.

Detailed description

Emerging evidence indicates that upregulation of the endoplasmic reticulum (ER) stress-induced pro-osteogenic transcription factor ATF4 (activating transcription factor 4) plays an important role in vascular calcification, a common complication in patients with aging, diabetes and chronic kidney disease (CKD).

Rosa26-ATF4^loxtg mutant mice carry a floxed stop/FLAG-tagged human ATF4 cassette knocked into the mouse Gt(ROSA)26Sor gene. Cre-mediated excision of the floxed stop enables site-directed expression of FLAG-tagged human ATF4 protein.

Progeny of Rosa26-ATF4^loxtg mice intercrossed with smooth muscle-specific SMMHC-Cre^ER(T2) animals (see Stock No. 007742) that are treated with tamoxifen to induce ATF4 expression display severe medial calcification. The aortic calcium content is increased by 2.5 fold, and increased numbers of apoptotic cells are seen in the aorta. Serum parameters including cholesterol, triglyceride, phosphorus and calcium are not altered.

Development

A loxP site, PGK-puromycin-pA cassette (in reverse transcriptional orientation), 4x SV40-pA Stop, loxP, and FLAG-human ATF4-WPRE-BGH-pA cassette sequences were introduced to the Gt(ROSA)26Sor gene via a FLP recombinase-mediated cassette exchange method.

The R26FNF3-1F1 (1F1) ES cell line used to generate the mutation was created by targeting Gt(ROSA)26Sor with an FRT- and F3-flanked PGK-neomycin selection cassette by homologous recombination in (C57BL/6J x 129X1/SvJ)F1-derived EC7.1 ES cells.

1F1 embryonic stem (ES) cells were transfected with plasmids pFLSLF2-FLAG human ATF4 (carrying FRT-loxP-Stop-loxP-FRT-FLAG-ATF4) and pCAG-Flpe (expressing FLP recombinase) to create the Rosa26-ATF4^loxtg mutation.

Resultant founder mice carrying a floxed stop/FLAG-tagged human ATF4 cassette were backcrossed to C57BL/6J for 10 generations by the donating laboratory (see SNP note below).

A 48 SNP (single nucleotide polymorphism) panel analysis, with 43 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. Two of the 43 markers throughout the genome are segregating, suggesting an incomplete backcross. Also, 1 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: Gt(ROSA)26Sor^tm2(ATF4)Myz
Name: targeted mutation 2, Makoto Miyazaki
MGI accession ID: MGI:5812282
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Inserted expressed sequence; Humanized sequence
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: (C57BL/6J x 129X1/SvJ)F1
Expressed genes: ATF4,activating transcription factor 4,human
Site of expression: Following Cre recombination, FLAG-tagged human ATF4 protein is expressed in Cre-expressing tissues.
Molecular note: The targeting vector containing a loxP site, PGK-puromycin-pA cassette (in reverse transcriptional orientation), 4x SV40-pA STOP, loxP, and FLAG-human ATF4-WPRE-BGH-pA cassette sequence is introduced to the Gt(ROSA)26Sor gene via a FLP recombinase-mediated cassette exchange method.