STOCK Plk4^em4Ahol/J

Stock number: 037681
Product name: Plk4^cKD
Research areas: Cancer Research, Cell Biology Research, Research Tools

Description

Plk4^cKD mice possess a loxP-STOP-loxP cassette in intron 4 followed by a kinase-inactivating point mutation (D154A) in exon 5 of the polo-like kinase 4 gene (Plk4). Removal of the cassette with Cre recombinase generates mice that express mutant (kinase-dead) PLK4. These mice may be useful for studying the role of Plk4 in centriole biology and centrosome loss in multiple disorders.

The donating laboratory also created a conditional wildtype strain, Plk4^cWT (Stock No. 037683), to be utilized as a control for this Plk4^cKD line.

Detailed description

Polo-like kinase 4 (PLK4) is a serine/threonine kinase that regulates duplication of centrioles during cell-cycle progression. Overexpression of PLK4 may result in centriole amplification which is a common feature of cancer cells and other chromosomal instability disorders, while PLK4 loss causes centrosome duplication failure. Plk4^cKD mice have loxP sites flanking a STOP cassette in intron 4 followed by a kinase-dead point mutation (D154A) in exon 5 of the polo-like kinase 4 gene (Plk4). When bred to a tissue-specific Cre recombinase strain, resulting offspring will express the kinase-inactivating point mutation D154A in cre-expressing tissues. This point mutation leads to accumulation of inactive PLK4 producing a loss-of-function phenotype. Prior to cre-recombination, wildtype PLK4 expression is disrupted, resulting in a null allele. The donating laboratory also created a conditional wildtype strain, Plk4^cWT (Stock No. 037683), to be utilized as a control for this Plk4^cKD line.

Mice that are homozygous for this floxed Plk4^cKD allele are embryonic lethal. These mice may be useful for studying the role of Plk4 in centriole biology and centrosome loss in multiple disorders.

The donating investigator has made multiple Plk4 mouse lines available: Plk4^Δu (Stock No. 037482), Plk4^Dox (Stock No. 037530), Plk4^f (Stock No. 037549) and Plk4^cWT (Stock No. 037683).

Development

The Plk4^cKD allele was generated using CRISPR/Cas9 endonuclease-mediated homology-directed repair (HDR) genome editing. Guide RNAs, targeting DNA sequence 5’-aagctaggactttaatactc-3’, were used to insert a loxP flanked STOP cassette into intron 4 followed by a kinase-dead point mutation (D154A) into exon 5 of the Plk4 gene. Plk4-201 was used as reference for the exon number and the guide sequences. A double-stranded DNA donor template along with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes were injected into the cytoplasm of zygotes derived from B6SJLF1 mice with well recognized pronuclei. Correctly targeted pups were identified by PCR and sequencing. The resulting Plk4^cKD mice were maintained by crossing to heterozygous Plk4^F (Stock No. 037549); heterozygous R26-CreERT2 (Stock No. 008463) double mutant by the donating laboratory. Prior to arrival at The Jackson Laboratory, the line was crossed to C57BL/6J mice to remove the Plk4^F and R26-CreERT2 alleles. Upon arrival, 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: Plk4^em4Ahol
Name: endonuclease-mediated mutation 4, Andrew Holland
MGI accession ID: MGI:7341824
Generation method: Endonuclease-mediated
Attributes: Conditional ready (e.g. floxed)
Synonyms: Plk4^cKD
Marker symbol: Plk4
Marker name: polo like kinase 4
Marker synonyms: MCCRP2; Sak; Stk18
Chromosome: 3
Strain of origin: (C57BL/6 x SJL/J)F1
Molecular note: CRISPR/Cas9 genome editing uses guide RNAs, targeting DNA sequence 5'-aagctaggactttaatactc-3';, are used to insert a loxP flanked STOP cassette into intron 4 followed by a kinase-dead point mutation (D154A) into exon 5. Plk4-201 was used as reference for the exon number and the guide sequences.