B6;129P2-Gt(ROSA)26Sor^{em2(CAG-LOC4337827*)Mbu}/J

Stock number: 039661
Product name: Rosa26(Tir1-F74G) 
Strain synonyms: Rosa26^Tir1-F74G , Rosa26^OsTir1-F74G , Rosa26^OsTir1*F74G
Research areas: Cell Biology Research, Research Tools

Description

Rosa26(Tir1-F74G) knock-in (KI) mice constitutively express a modified 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.

The donating laboratory has made other mouse lines available for use in the AID2 system: Pax5^AID Rosa26^Tir1-F74G (Stock No. 040293), Ebf1^AID Rosa26^Tir1-F74G (Stock No. 040294), Tcf3^AID Rosa26^Tir1-F74G (Stock No. 040295), Ikzf1^AID Ikzf3^AID Rosa26^Tir1-F74G (Stock No. 040296), Ikzf1^AID Rosa26^Tir1-F74G (Stock No. 040297), Ikzf3^AID Rosa26^Tir1-F74G (Stock No. 040298), Prdm1^AID Rosa26^Tir1-F74G (Stock No. 040299) and Tcf4^AID Rosa26^Tir1-F74G (Stock No. 040300).

Detailed description

Rosa26(Tir1-F74G) knock-in (KI) mice were developed to constitutively 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; the F74G mutation abolishes the spontaneous degradation activity of TIR1 and increases the affinity for the auxin derivative 5-phenyl-indole-3-acetic acid (5-Ph-IAA). Following addition of 5-Ph-IAA, the AID-tagged protein is degraded by the proteasome. In these KI mice, a CAG promoter directs expression of the TIR1^F74G sequence, linked to an N-terminal nuclear localization signal (NLS), from the Gt(ROSA)26Sor locus. Homozygous Rosa26(Tir1-F74G) KI mice are viable and fertile. These mice may be a valuable tool for studying protein function in vivo.

The donating laboratory has made other mouse lines available for use in the AID2 system: Pax5^AID Rosa26^Tir1-F74G (Stock No. 040293), Ebf1^AID Rosa26^Tir1-F74G (Stock No. 040294), Tcf3^AID Rosa26^Tir1-F74G (Stock No. 040295), Ikzf1^AID Ikzf3^AID Rosa26^Tir1-F74G (Stock No. 040296), Ikzf1^AID Rosa26^Tir1-F74G (Stock No. 040297), Ikzf3^AID Rosa26^Tir1-F74G (Stock No. 040298), Prdm1^AID Rosa26^Tir1-F74G (Stock No. 040299) and Tcf4^AID Rosa26^Tir1-F74G (Stock No. 040300).

Development

The Rosa26(Tir1-F74G) knock-in (KI) allele was generated by introducing the F74G mutation into an existing Rosa26^Tir1 allele (Baker et al, Sci. Rep, 2016), using CRISPR/Cas9 endonuclease-mediated homology-directed repair (HDR) genome editing.

Dr. Meinrad Busslinger acquired the Rosa26^Tir1 mouse model, on a C57BL/6 background, from Dr. A Francis Stewart [Dresden University of Technology, Germany]. In brief, the Rosa26^Tir1 target vector was designed to insert a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), an Oryza sativa sequence, linked to an N-terminal nuclear localization signal (NLS), an internal ribosome entry site (IRES) sequence and a puromycin cassette, into intron 1 of the Gt(ROSA)26Sor locus on chromosome 6. The construct was electroporated into 129P2/OlaHsd-derived E14TG2a embryonic stem (ES) cells and correctly targeted ES cells were injected into recipient blastocysts. The resulting chimeric mice were backcrossed onto a C57BL/6 background for an unknown number of generations. The Rosa26^Tir1 mice were further used as described below.

To introduce the F74G modification, a single guide RNA [5'-AAATTGAAATCAGCAAAATG-3'], a 200-bp single-stranded repair DNA oligonucleotide and Cas9 protein were introduced into homozygous Rosa26^Tir1 zygotes, on a C57BL/6 background, and transferred to pseudopregnant females. Correctly targeted pups were identified by PCR and sequencing. The resulting heterozygous Rosa26(Tir1-F74G) KI mice were backcrossed to C57BL/6J mice for another 3 generations by the Busslinger 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^{em2(CAG-LOC4337827*)Mbu}
Name: endonuclease-mediated mutation 2, Meinrad Busslinger
MGI accession ID: MGI:7703958
Generation method: Endonuclease-mediated
Attributes: Inserted expressed sequence
Synonyms: Rosa26^Tir1-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: 129P2/OlaHsd
Molecular note: The CAG promoter drives a codon-optimized Oryza sativa F-box transport inhibitor response 1 (TIR1) sequence, linked to an N-terminal nuclear localization signal (NLS), an internal ribosome entry site (IRES) sequence and a puromycin cassette, inserted into intron 1 of the Gt(ROSA)26Sor locus. The allele was generated by introducing the F74G mutation into an existing Rosa26(Tir1) allele CRISPR-Cas9 methodology introduced the F74G mutation into an existing F-box transport inhibitor response 1 (TIR1) protein carried by the Gt(ROSA)26Sor^{tm2(CAG-LOC4337827)Afst} allele.