B6;SJL-Plk4^em1Ahol/J

Stock number: 037482
Product name: Plk4^Δu
Research areas: Cancer Research, Cell Biology Research

Description

Plk4 ^Δu mice carry a CRISPR/Cas9-made mutation that knocks out start codons of the Plk4 upstream open reading frames uORF1 and uORF2. These mice may be useful for studying the role of Plk4 in centriole biology, regulation of gametogenesis and centrosome amplification in multiple cancers.

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. The donating laboratory has determined that upstream open reading frames (uORF) in the 5’UTR of the Plk4 gene are responsible for regulating translation of PLK4 mRNA. Plk4 ^Δu mice possess a CRISPR/Cas9-made mutation that knocks out start codons of the Plk4 upstream open reading frames uORF1 and uORF2. This mutation results in increased expression of PLK4, specifically in primordial germ cells (PGCs), which promotes centriole amplification and cell division errors that ultimately drive cell death. Homozygous mice are viable but display dramatically reduced fertility because of a significant depletion of PGCs. These Plk4^Δu mice may be useful for studying the role of Plk4 in centriole biology and regulation of gametogenesis.

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

Development

The Plk4 ^Δu allele was generated using CRISPR/Cas9 endonuclease-mediated homology-directed repair (HDR) genome editing. Two guide RNAs (uORF1 sgRNA: 5'- CCGGCGGGAATTTTTCAAAA-3’ and uORF2 sgRNA: 5'- GCGTAGAGAAGGCGTCCTGA-3') and a single-stranded DNA repair template containing the desired modification (ATG start codons of uORF1 and uORF2 were changed to ATA). Plk4 transcript Plk4-201 was used as reference for the exon number, and the guide and donor sequences. sgRNAs, ssDNA, and the Cas9 nuclease mRNA were introduced into the cytoplasm of zygotes derived from B6SJLF2 mice with well recognized pronuclei. Correctly targeted pups were identified by PCR and sequencing. The resulting Plk4 ^Δu/+ mice were backcrossed to C57BL/6J mice for one generation to minimize off-target CRISPR effects. The donating investigator reports their colony exhibits white/albino, agouti and black coat colors (as expected for a mixed C57BL/6;SJL genetic background). 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: Plk4^em1Ahol
Name: endonuclease-mediated mutation 1, Andrew Holland
MGI accession ID: MGI:7316773
Generation method: Endonuclease-mediated
Attributes: Not Applicable
Marker symbol: Plk4
Marker name: polo like kinase 4
Chromosome: 3
Strain of origin: (C57BL/6 x SJL)F2
Molecular note: CRISPR/cas9 genome editing uses two guide RNAs (uORF1 sgRNA: 5'- CCGGCGGGAATTTTTCAAAA-3' and uORF2 sgRNA: 5'- GCGTAGAGAAGGCGTCCTGA-3') and a single-stranded DNA repair template to insert the desired modification (ATG start codons of uORF1 and uORF2 were changed to ATA).