B6.Cg-Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} Col1a1^{tm1(tetO-Plk4,-EYFP)Ahol}/J

Stock number: 037530
Product name: Plk4^Dox
Research areas: Cancer Research, Cell Biology Research, Research Tools

Description

Plk4^Dox mice may be used to induce overexpression of Plk4 via a tetracycline-controlled transactivator protein system. These mice may be useful for studying the role of Plk4 in centriole biology 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 centrosome amplification which is a common feature of cancer cells and may promote chromosomal instability disorders. The Plk4^Dox mouse model may be utilized to evaluate the effect of Plk4 overexpression in a controlled manner, using the tet-responsive element (tetO). This strain also contains the optimized form of reverse tetracycline controlled transactivator (rtTA-M2). Increased expression of Plk4 can be regulated with the tetracycline analog doxycycline (dox) in these double mutant offspring. Using this inducible mouse model, the donating laboratory found significant increases in Plk4 mRNA levels across multiple tissues, with exception of the brain, after doxycycline exposure. They also found that increases in PLK4, after 1 or 8-months of continuous doxycycline treatment, promotes aneuploidy and centrosome amplification leading to development of spontaneous tumors across multiple tissues. These Plk4^Dox mice may be useful for studying the role of Plk4 in centriole biology and centriole amplification in tumor development.

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

A targeting vector was designed to insert frt-flanked PGK-Neo-pA 500 bp downstream of the 3' UTR of the Col1a1 locus. This construct was electroporated into (C57BL/6 x 129S4Sv/Jae)F1-derived V6.5 embryonic stem (ES) cells. Correctly targeted ES cells were identified. One of these ES cells clones was re-targeted to insert an optimized form of reverse tetracycline controlled transactivator (rtTA-M2) followed by a rabbit beta-globin intron/polyA signal and PGK-puromycin cassette, all downstream of the Gt(ROSA)26Sor promoter. Correctly targeted ES cells were identified. One of these ES cell clones, KH2, was selected and the frt-flanked PGK-Neo-pA in the 3' UTR of the Col1a1 locus was replaced by co-injection of a tetO-Plk4-EYFP plasmid and flpe recombinase. The Plk4-EYFP plasmid contained mouse Plk4 open reading frame (ORF) C-terminally tagged with EYFP downstream of the tetracycline response element (tetO) and CMV minimal promoter.

The resulting ES cells, now targeted with rtTA-M2 in the Gt(ROSA)26Sor locus (Rosa26-rtTA) and tetO-Plk4-EYFP in the Col1a1 locus, were injected into recipient blastocysts. The resulting chimeric mice were bred with C57BL/6N mice and later backcrossed to C57BL/6J (Stock No. 000664) mice for 8 generations by donating laboratory (see SNP note below). Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes. Of note, the donating investigator indicated that the Y chromosome was fixed on the C57BL/6J background.

In 2022, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the males sent to The Jackson Laboratory Repository. One of the 43 markers throughout the genome were segregating with 129 on Chromosome 9. Also, all two of the five markers that determine C57BL/6J from C57BL/6N were found to be segregating, including the rd8 recessive mutation associated with retinal degeneration on Chromosome 1 within the Crb1 locus. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6N;C57BL/6J genetic background.

Allele

Symbol: Col1a1^{tm1(tetO-Plk4,-EYFP)Ahol}
Name: targeted mutation 1, Andrew Holland
MGI accession ID: MGI:7316646
Generation method: Targeted
Attributes: Reporter; Inducible; Inserted expressed sequence
Marker symbol: Col1a1
Marker name: collagen, type I, alpha 1
Chromosome: 11
Strain of origin: (C57BL/6 x 129S4/SvJae)F1
Molecular note: A targeting vector is designed to insert FRT-flanked PGK-Neo-promoterless, ATG-less hygromycin cassette 500 bp downstream of the 3' UTR of the Col1a1 locus. Flpe recombinase is then used to FLP-IN a tetO-Plk4-EYFP plasmid. The Plk4-EYFP plasmid contained mouse Plk4 open reading frame (ORF) C-terminally tagged with EYFP downstream of the tetracycline response element (tetO) and CMV minimal promoter.

Allele

Symbol: Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae}
Name: targeted mutation 1, Rudolf Jaenisch
MGI accession ID: MGI:3702294
Generation method: Targeted
Attributes: Transactivator
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: (C57BL/6 x 129S4/SvJae)F1
Expressed genes: rtTA,reverse tetracycline-controlled transactivator,E. coli
Site of expression: Expresses an optimized rtTA protein (rtTA-M2). Inducible target gene expression is detected in liver, bone marrow, stomach, intestine, and skin, with lower levels in the heart, lungs, kidney, spleen, and thymus; no expression is detected in the brain and testes.
Molecular note: An optimized form of reverse tetracycline controlled transactivator (rtTA-M2) was inserted downstream of the Gt(ROSA)26Sor promoter and was followed by a PGK-puro selection cassette. This mutant form of rtTA termed M2 has five amino acid substitutions in the tetR moiety of tTA: S12G, E19G, A56P, D148E and H179R. This mutated form of transactivator protein has increased doxycycline sensitivity. Mice have widespread expression of the rtTA-M2 protein.
General note: This mutation was created during the generation of mutant ES cell line KH2, which also contains a frt docking site downstream of the endogenous Col1a1 locus on Chr 11 (Col1a1). The two mutations have subsequently been bred apart.