B6;SJL-Cep192^{em1(mNeonGreen*)Ahol}/J

Stock number: 039573
Product name: Cep192^{mAID-mNeonGreen}
Strain synonyms: Cep192^AID
Research areas: Cell Biology Research, Research Tools

Description

Cep192^{mAID-mNeonGreen} knock-in (KI) mice express a centrosomal protein 192 (Cep192) mutation where the C-terminal exon is tagged with a miniaturized auxin-inducible degradation-promoting sequence (degron) tag (mAID) and mNeonGreen. In conjunction with an OsTir1-F74G mouse line (e.g., Stock No. 039572), these mice may be useful for studying the role of CEP192 protein in centrosome biology and mitosis.

Detailed description

Cep192 encodes a protein required for mitotic centrosome and spindle assembly. These Cep192^{mAID-mNeonGreen} knock-in (KI) mice express endogenous centrosomal protein 192 (Cep192) where the C-terminal exon is tagged with a miniaturized AID tag (mAID) and mNeonGreen. In conjunction with the conditional OsTir1-F74G mouse line (Stock No. 039572), and treatment with the auxin derivative 5-Ph-IAA, resulting progeny will have rapid, reversible degradation of CEP192 protein in cre-expressing tissues. Degradation of the CEP192 protein may be measured using direct fluorescence imaging of mNeonGreen.

Homozygous Cep192^{mAID-mNeonGreen} KI mice are viable and fertile with no overt phenotype. The donating laboratory evaluated the second generation auxin-inducible-degron (AID2) technology by first crossing the conditional OsTir1-F74G mouse line (Stock No. 039572) to a Sox2-Cre mouse line (Stock No. 008454) to generate progeny with ubiquitous expression of OsTir1-F74G (OsTir1^Tir). The OsTir1^Tir mice were then bred to Cep192^{mAID-mNeonGreen} mice and resulting progeny were treated with 5mg/kg of the auxin derivative 5-Ph-IAA. CEP192 protein degradation was detectable within 30 minutes of the treatment across multiple tissues with protein levels remaining low for 24 hours after administration. CEP192 protein levels did show a complete recovery 72 hours post-injection. These Cep192^{mAID-mNeonGreen} mice may be useful for research on centrosome biology and mitosis.

Development

The Cep192^{mAID-mNeonGreen} knock-in (KI) allele was generated using CRISPR/Cas9 endonuclease-mediated homology-directed repair (HDR) genome editing. A single guide RNA [5'-cgactaatcggtgaatccct-3'] was used to introduce a flexible linker, mAID [miniaturized auxin-inducible degradation-promoting sequence (degron) tag] and moxNeonGreen sequence into exon 48 of the centrosomal protein 192 (Cep192) gene on chromosome 18. 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 Cep192^{mAID-mNeonGreen} KI mice were backcrossed to C57BL/6 mice for at least 2 generations 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: Cep192^{em1(mNeonGreen*)Ahol}
Name: endonuclease-mediated mutation 1, Andrew Holland
MGI accession ID: MGI:7657344
Generation method: Endonuclease-mediated
Attributes: Reporter; Inducible degradation
Synonyms: Cep192^{mAID-mNeonGreen}
Marker symbol: Cep192
Marker name: centrosomal protein 192
Marker synonyms: 4631422C13Rik; D430014P18Rik; Da1-6; PPP1R62; Seh1l
Chromosome: 18
Strain of origin: (C57BL/6 x SJL)F2
Molecular note: Using CRISPR/Cas9 technology, a single guide RNA [5'-cgactaatcggtgaatccct-3'] was used to introduce a flexible linker, mAID [miniaturized auxin-inducible degradation-promoting sequence (degron) tag] and moxNeonGreen sequence into exon 48 of the centrosomal protein 192 (Cep192) gene on chromosome 18.