B6.129P2(SJL)-Ttk^tm2.1Kops/JlmaJ

Stock number: 035137
Product name: CiMKi^KD
Research areas: Cell Biology Research, Research Tools

Description

Cre-inducible-Mps1-Knock-in-KD (CiMKi^KD) mice carry a Cre-inducible point mutation (D637A) in exon 17 of the Ttk protein kinase (Ttk) gene, expression of which is blocked by the presence of a loxP-flanked cDNA cassette encoding exons 17-22 and a stop codon. This mutation results in a kinase dead protein similar to that found in human cell lines with a severe chromosomal instability (CIN) phenotype. This strain may be useful when studying oncogenic potential due to the degree and site of chromosomal instability.

Detailed description

The Ttk protein kinase gene, Ttk, also known as Mps1 (Monopolar spindle 1 kinase) encodes a protein kinase with the ability to phosphorylate tyrosine, serine, and threonine. It is an essential spindle assembly checkpoint protein, and critical for chromosome alignment at the centromere during mitosis, and is required for centrosome duplication. Deregulation of TTK protein or its kinase activity, results in aneuploidy due to chromosomal instability (CIN). CIN and aneuploidy are associated with tumor aggressiveness, therapy resistance, and poor patient prognosis.

Cre-inducible-Mps1-Knock-in-KD (CiMKi^KD) mice have a loxP-flanked cassette containing cDNA encoding exons 17-22 from the Ttk gene inserted downstream of the endogenous exon 16. The 3' loxP site is preceded by a stop codon and polyadenylation sequence. Downstream of this cassette, endogenous exon 17 was modified to harbor a GAT to GCT mutation at codon 637, resulting in an aspartic acid to alanine mutation (D637A) that causes complete loss of Mps1 kinase activity. Expression of the mutant D637A protein is achieved by Cre-mediated removal of the cassette containing exons 17-22 and the associated polyadenylation sequence. Mice that are heterozygous and homozygous for this allele are viable, while some homozygous mice show a decrease in fertility perhaps correlating with external stress.

When bred to mice that express tissue-specific Cre recombinase, resulting offspring will have the floxed endogenous exons 17-22 removed and the D637A mutant exon 17 placed in transcriptional orientation.

Of note, CiMKi^TA mice are similar, expressing a milder CIN phenotype after Cre recombination, and are available as Stock No. 035136. This strain contains a similar construct, with endogenous exon 17 modified to harbor an ACA to GCA mutation at codon 649, resulting in a threonine to alanine mutation (T649A) that causes reduced kinase activity of Mps1.

When these CiMKi^KD mice are used in combination with CiMKi^TA and wild-type Mps1 (WT) mice, five different levels of CIN can be induced: very low (WT/TA), low (WT/KD), moderate (TA/TA), high (TA/KD), and very high (KD/KD).

For example, when bred to Apc^Min mice (Stock No. 002020), which are predisposed to intestinal cancer, and CiMKi^TA mice, moderate CIN causes an increase in adenoma burden in the large intestines and in the distal colon, with no effect in the small intestine.

Development

The CiMKi^KD targeting vector was designed to insert loxP-flanked cassette containing cDNA encoding exons 17-22 from Ttk protein kinase (Ttk) gene downstream of the endogenous exon 16. The 3' loxP site was preceded by a stop codon and polyadenylation sequence. Downstream of this cassette, endogenous exon 17 was modified to harbor a GAT to GCT mutation at codon 637, resulting in an aspartic acid to alanine mutation (D637A) similar to that found in human cell lines with a severe chromosomal instability (CIN) phenotype. A frt-flanked puromycin cassette was also included. The construct was electroporated into 129P2/OlaHsd-derived IB10 embryonic stem (ES) cells. ES cells were injected into C57BL/6 blastocysts and resulting chimeric mice were mated to C57BL/6J. Resulting offspring were bred to ACTB:FLPe mice (Stock No. 005703) to remove the selection cassette. Resulting CiMKi^KD mice were bred together to remove the Flpe expressing transgene, and offspring were bred to C57BL/6J mice for at least 7 generations. Upon arrival at The Jackson Laboratory Repository, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

In December 2020, The Jackson Laboratory sequencing results identified the expected mutation (D637A) in exon 17, as well as a single nucleotide difference located 19 bp 5' upstream of exon 17 (1 bp insertion of an adenosine base in intron 16). This corresponds to SNP variant rs219488405 (the A insertion associated with 129 genome). While it has not been specifically determined if this has any effect on splicing efficiency, its position relative to exon 17 makes it unlikely to have any impact on splicing efficiency - see our sequencing assay.

Allele

Symbol: Ttk^tm2.1Kops
Name: targeted mutation 2.1, Geert Kops
MGI accession ID: MGI:6404036
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Humanized sequence
Synonyms: CiMKi^KD
Marker symbol: Ttk
Marker name: Ttk protein kinase
Marker synonyms: CT96; ESK; Esk1; MPH1; Mps1; MPS1L1; PYT
Chromosome: 9
Strain of origin: 129P2/OlaHsd
Molecular note: The targeting vector was designed to insert a loxP-flanked cassette containing cDNA encoding exons 17-22 from the gene downstream of the endogenous exon 16. The 3' loxP site was preceded by a stop codon and polyadenylation sequence. Downstream of this cassette, endogenous exon 17 was modified to harbor a GAT to GCT mutation at codon 637, resulting in an aspartic acid to alanine mutation (D637A) similar to that found in human cell lines with a mild chromosomal instability (CIN) phenotype. An FRT-flanked puromycin cassette was also included and then removed by Flp-mediated recombination.