B6;SJL-Gt(ROSA)26Sor^{em1(CAG-LOC4337827*)Ahol}/J

Stock number: 039572
Product name: Rosa26-OsTir1-F74G^flox
Strain synonyms: OSTir1^Flox, Rosa26^{LSL-OSTir1-F74G} , Rosa26^{LSL-OSTir1*F74G}
Research areas: Cell Biology Research, Research Tools

Description

Rosa26-OsTir1-F74G^flox mice express a Cre-inducible Oryza sativa F-box transport inhibitor response 1 protein, harboring the F74G modification, for use in the second generation auxin-inducible degron (AID2) system resulting in rapid and reversible degradation of auxin inducible degron-tagged proteins in cre-expressing cells.

Detailed description

Rosa26-OsTir1-F74G^flox knock-in (KI) mice were developed to express the Japanese rice Oryza sativa F-box transport inhibitor response 1 (TIR1, formal name LOC4337827) protein, harboring the F74G modification, for use in the second generation auxin-inducible degron (AID2) system for rapid and reversible degradation of auxin inducible degron-tagged (AID) proteins. TIR1 interacts with the mouse Cullin-RING ubiquitin ligase complex and an AID-targeted protein. Following addition of the auxin derivative 5-Ph-IAA, the target protein is degraded by the proteasome. A CAG promoter directs expression of a c-Myc (Myc) tagged TIR1^F74G sequence from the Gt(ROSA)26Sor locus.

Homozygous Rosa26-OsTir1-F74G^flox mice are viable and fertile. When bred to mice that express tissue-specific Cre recombinase, resulting offspring will have TIR1-F74G expressed in the cre-expressing tissues. The donating laboratory confirmed expression of TIR1-F74G across various tissues, after crossing these mice to a Sox2Cre mouse line (Stock No. 008454), with the lowest expression detected in the brain and kidney. These mice may be a valuable tool for studying protein function.

Development

The Rosa26-OsTir1-F74G^flox knock-in (KI) allele was generated using CRISPR/Cas9 endonuclease-mediated homology-directed repair (HDR) genome editing. A single guide RNA [5'-actccagtctttctagaaga-3'] was used to introduce a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), a loxP-flanked STOP cassette including a puromycin cassette, the codon-optimized Oryza sativa sequence harboring the F74G modification and a c-myc (Myc) epitope tag, into intron 1 of the Gt(ROSA)26Sor locus on chromosome 6. The guide RNA, donor sequence and Cas9 nuclease were introduced into B6SJLF2 zygotes and transferred to pseudopregnant females. Correctly targeted pups were identified by PCR and sequencing. The resulting heterozygous Rosa26-OsTir1-F74G^flox KI mice were backcrossed to C57BL/6 mice for at least 1 generation by the donating laboratory. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Gt(ROSA)26Sor^{em1(CAG-LOC4337827*)Ahol}
Name: endonuclease-mediated mutation 1, Andrew Holland
MGI accession ID: MGI:7657342
Generation method: Endonuclease-mediated
Attributes: Conditional ready (e.g. floxed); Inserted expressed sequence; Epitope tag
Synonyms: Rosa26^{LSL-OSTir1-F74G}
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: (C57BL/6 x SJL)F2
Molecular note: Using CRISPR/Cas9 technology, single guide RNA [5'-actccagtctttctagaaga-3'] was used to introduce a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), a loxP-flanked STOP cassette including a puromycin cassette, the codon-optimized Oryza sativa F-box transport inhibitor response 1 (TIR1) sequence harboring the F74G modification and a c-myc (Myc) epitope tag, into intron 1 of the Gt(ROSA)26Sor locus on chromosome 6.