B6.Cg-Pax5^em1Mbu Gt(ROSA)26Sor^{em2(CAG-LOC4337827*)Mbu}/J

Stock number: 040293
Product name: Pax5^Aid Rosa26^Tir1-F74G
Research areas: Cell Biology Research, Hematological Research, Immunology, Inflammation and Autoimmunity Research, Research Tools

Description

Pax5^AID Rosa26^Tir1-F74G 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 Pax5 allele. This mouse line can be used in the second-generation auxin-inducible degron (AID2) system, resulting in rapid and reversible degradation of PAX5-AID tagged protein in the B-cell and neuronal lineages.

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

This Pax5^Aid Rosa26^Tir1-F74G mouse line was generated by crossing Rosa26^Tir1-F74G mice (Stock No. 039661) with mice that express an auxin-inducible degron (AID)-tagged Pax5 knock-in allele (Pax5^AID). The resulting Pax5^AIDRosa26^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 paired box 5 (Pax5) gene. TIR1 interacts with the mouse Cullin-RING ubiquitin ligase complex and the AID-targeted PAX5 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), which as intraperitoneally injected at 10mg/kg body weight. Following addition of 5-Ph-IAA, the C-terminal AID-tagged PAX5 is degraded by the proteasome.

Mice that are homozygous for both alleles are viable and fertile with no overt phenotype. As Pax5 codes for a transcription factor essential for B-cell differentiation and neuronal development, the donating laboratory measured the frequencies of pro-B, pre-B and immature B cells isolated from Pax5^Aid Rosa26^Tir1-F74G bone marrow via flow cytometry. Prior to 5-Ph-IAA exposure, B cell lymphopoiesis was similar to controls; however, after 3 consecutive days of 5-Ph-IAA injections, pro-B, pre-B and immature B cells were notably decreased. These Pax5^Aid Rosa26^Tir1-F74G mice may be useful for studying the role of the PAX5 protein in B-cell development and neural differentiation.

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

This Pax5^Aid Rosa26^Tir1-F74G mouse line was generated by crossing Rosa26^Tir1-F74G mice (Stock No. 039661) with mice that express an auxin-inducible degron (AID)-tagged Pax5 knock-in allele (Pax5^AID).

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 Pax5^Aid allele was generated using CRISPR/Cas9 endonuclease-mediated genome editing. A single guide RNA (5'-GGGTCAGTGACGGTCATAGG-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 paired box 5 (Pax5) gene on chromosome 4. Pax5 transcript Pax5-201 (ENSMUST00000014174.14) 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 Pax5^AID mice were backcrossed to C57BL/6J mice for at least 6 generations before crossing to Rosa26^Tir1-F74G mice (JAX Stock No. 039661) to generate the Pax5^Aid Rosa26^Tir1-F74G mouse line. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish a live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J (Stock No. 000664) oocytes.

Allele

Symbol: Pax5^em1Mbu
Name: endonuclease-mediated mutation 1, Meinrad Busslinger
MGI accession ID: MGI:7869361
Generation method: Endonuclease-mediated
Attributes: Epitope tag; Inducible degradation
Synonyms: Pax5^Aid
Marker symbol: Pax5
Marker name: paired box 5
Marker synonyms: ALL3; BSAP; EBB-1; PAX-5; RGD1565236
Chromosome: 4
Strain of origin: (C57BL/6J x CBA/J)F1
Molecular note: A single guide RNA (5'-GGGTCAGTGACGGTCATAGG-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 paired box 5 (Pax5) gene on chromosome 4.

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.