B6.Cg-Gt(ROSA)26Sor^{em2(CAG-LOC4337827*)Mbu} Ikzf1^em1Mbu Ikzf3^em1Mbu/J

Stock number: 040296
Product name: Ikzf1^Aid Ikzf3^Aid Rosa26^Tir1-F74G
Research areas: Cell Biology Research, Hematological Research, Immunology, Inflammation and Autoimmunity Research, Research Tools

Description

Ikzf1^AID Ikzf3^AID Rosa26^Tir1-F74G triple mutant mice constitutively express a modified Oryza sativa F-box transport inhibitor response 1 protein, harboring the F74G modification, as well as a C-terminal AID-tagged Ikzf1 allele and a C-terminal AID-tagged Ikzf3 allele. This mouse line can be used in the second-generation auxin-inducible degron (AID2) system, resulting in rapid and reversible degradation of Ikaros (IKZF1)-AID tagged protein and Aiolos (IKZF3)-AID tagged protein in B-cell, T-cell and other immune cell lineages.

The donating laboratory has made other mouse lines available for use in the AID2 system: Rosa26^Tir1-F74G (Stock No. 039661), 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 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

This Ikzf1^Aid Ikzf3^Aid Rosa26^Tir1-F74G triple mutant mouse line was generated by crossing Rosa26^Tir1-F74G mice (Stock No. 039661) with mice that express an auxin-inducible degron (AID)-tagged Ikzf1 knock-in allele (Stock No. 040297) and then with mice that express an AID-tagged Ikzf3 knock-in allele (Stock No. 040298). The resulting Ikzf1^AID Ikzf3^AID Rosa26^Tir1-F74G mice 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. This strain also has a glycine linker, a minimal auxin-inducible degron (Aid) sequence and the V5 epitope, inserted immediately upstream of the 3' UTR of the IKAROS family zinc finger 3 (Ikzf3) gene of the IKAROS family zinc finger 1 (Ikzf1) gene, as well as a second glycine linker, Aid sequence and V5 epitope inserted immediately upstream of the 3' UTR of the IKAROS family zinc finger 3 (Ikzf3) gene. TIR1 interacts with the mouse Cullin-RING ubiquitin ligase complex and the AID-tagged Ikaros (IKZF1) and AID-tagged Aiolos (IKZF3) proteins; 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), which was intraperitoneally injected at 10 mg/kg body weight. Following addition of 5-Ph-IAA, the C-terminal AID-tagged Ikaros (IKZF1) and Aiolos (IKZF3) are degraded by the proteasome.

Mice that are homozygous for all three alleles are viable and fertile with no overt phenotype. Both Ikzf1 and Ikzf3 code for zinc-finger transcription factors required for B-cell and T-cell development and differentiation. The donating laboratory measured the frequencies of pro-B, pre-B and immature B cells isolated from Ikzf1^Aid Ikzf3^Aid Rosa26^Tir1-F74G bone marrow via flow cytometry. Prior to 5-Ph-IAA exposure, B cell lymphopoiesis was similar to controls; however, after 6 consecutive days of 5-Ph-IAA injections, immature B cells and small pre-B cells were completely lost on simultaneous degradation of the Ikaros (IKZF1) and Aiolos (IKZF3) proteins. These Ikzf1^Aid Ikzf3^Aid Rosa26^Tir1-F74G triple mutant mice may be useful for studying the roles of Ikaros (IKZF1) and Aiolos (IKZF3) proteins in B-cell, T-cell and other immune cell development and differentiation.

The donating laboratory has made other mouse lines available for use in the AID2 system: Rosa26^Tir1-F74G (Stock No. 039661), 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 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

This Ikzf1^Aid Ikzf3^Aid Rosa26^Tir1-F74G triple mutant mouse line was generated by crossing Rosa26^Tir1-F74G mice (Stock No. 039661) with mice that express an auxin-inducible degron (AID)-tagged Ikzf1 knock-in allele (Stock No. 040297) and then with mice that express an AID-tagged Ikzf3 knock-in allele (Stock No. 040298).

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. See Stock No. 039661 for specific details about the development of this Rosa26^Tir1-F74G KI allele.

The Ikzf1^Aid allele was generated using CRISPR/Cas9 endonuclease-mediated genome editing. A single guide RNA (5'-GGCCTGGCTGGGTTTAGCTC-3') was used to insert a glycine linker, a minimal auxin-inducible degron sequence and the V5 epitope in frame to the last amino acid sequence of the IKAROS family zinc finger 1 (Ikzf1) gene on chromosome 11. Ikzf1 transcript Ikzf1-201 (ENSMUST00000018798.7) was used as reference for the exon number and the guide sequences. The sgRNAs, ssDNA, and the Cas9 nuclease were introduced into the cytoplasm of zygotes derived from (C57BL/6J x CBA/J)F1 mice with well recognized pronuclei. Correctly targeted pups were identified by PCR and sequencing. The resulting Ikzf1^AID mice were backcrossed to C57BL/6J mice for at least 6 generations before crossing to Rosa26^Tir1-F74G mice (Stock No. 039661) to generate the Ikzf1^Aid Rosa26^Tir1-F74G mouse line. These double mutant mice were then bred as described below.

The Ikzf3^Aid allele was generated using CRISPR/Cas9 endonuclease-mediated genome editing. A single guide RNA (5'-GACAGATGCTCACTTCAACA-3') was used to insert a glycine linker, a minimal auxin-inducible degron sequence and the V5 epitope in frame to the last amino acid sequence of the IKAROS family zinc finger 3 (Ikzf3) gene on chromosome 11. Ikzf3 transcript Ikzf3-201 (ENSMUST00000103141.4) was used as reference for the exon number and the guide sequences. The sgRNAs, ssDNA, and the Cas9 nuclease were introduced into the cytoplasm of zygotes derived from (C57BL/6J x CBA/J)F1 mice with well recognized pronuclei. Correctly targeted pups were identified by PCR and sequencing. The resulting Ikzf3^AID mice were backcrossed to C57BL/6J mice for at least 6 generations before crossing to Rosa26^Tir1-F74G mice (Stock No. 039661) to generate the Ikzf3^Aid Rosa26^Tir1-F74G mouse line. These double mutant mice were then bred to the Ikzf1^Aid Rosa26^Tir1-F74G mouse line (Stock No. 040297) to generate the triple mutant mouse line, referred to as Ikzf1^Aid Ikzf3^Aid Rosa26^Tir1-F74G (Stock No. 040296). Upon arrival at The Jackson Laboratory, sperm that was homozygous for all three (3) alleles was cryopreserved. To establish a live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J (Stock No. 000664) oocytes.

Allele

Symbol: Ikzf1^em1Mbu
Name: endonuclease-mediated mutation 1, Meinrad Busslinger
MGI accession ID: MGI:7869373
Generation method: Endonuclease-mediated
Attributes: Epitope tag; Inducible degradation
Synonyms: Ikzf1^AID
Marker symbol: Ikzf1
Marker name: IKAROS family zinc finger 1
Marker synonyms: 5832432G11Rik; CVID13; Hs.54452; IK1; Ikaros; LYF1; LyF-1; PPP1R92; PRO0758; RGD1562979; Zfpn1a1; ZNFN1A1
Chromosome: 11
Strain of origin: (C57BL/6J x CBA/J)F1
Molecular note: A single guide RNA (5'-GGCCTGGCTGGGTTTAGCTC-3') was used to insert a glycine linker, a minimal auxin-inducible degron sequence and the V5 epitope in frame to the last amino acid sequence of the IKAROS family zinc finger 1 (Ikzf1) gene on chromosome 11.

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.

Allele

Symbol: Ikzf3^em1Mbu
Name: endonuclease-mediated mutation 1, Meinrad Busslinger
MGI accession ID: MGI:7869376
Generation method: Endonuclease-mediated
Attributes: Epitope tag; Inducible degradation
Synonyms: Ikzf3^AID
Marker symbol: Ikzf3
Marker name: IKAROS family zinc finger 3
Marker synonyms: 5830411O07Rik; AIO; Aiolos; IMD84; Zfpn1a3; ZNFN1A3
Chromosome: 11
Strain of origin: (C57BL/6J x CBA/J)F1
Molecular note: A single guide RNA (5'-GACAGATGCTCACTTCAACA-3') was used to insert a glycine linker, a minimal auxin-inducible degron sequence and the V5 epitope in frame to the last amino acid sequence of the IKAROS family zinc finger 3 (Ikzf3) gene on chromosome 11.