Chromosome segregation errors: a double-edged sword - TI Pharma

activity by ~80% 170-172 . With the use of these two different Mps1 mutations we create a panel
of mice with graded reductions in Mps1 kinase activity (100%, 60%, 50%, 20%, 10% and 0%) and
as a result with various levels of CIN (Fig. 1). This way we will be able to test the hypothesis that
different levels of CIN will have different effects on tumor-initiation, -enhancement or –inhibition.
Using homologous recombination in mouse embryonic stem cells (ES cells) we have inserted a cDNA
sequence encompassing exons 17-22 followed by the polyA sequence and stop codon of Mps1 mRNA,
into intron 16 of the genomic sequence coding for Mps1 (Fig.2A). This results in transcription of
wild-type Mps1, but with the sequences of exons 17-22 being transcribed from the inserted intronless
cDNA. The inserted cDNA is flanked by LoxP sites, and the targeting vector included a right
A
9,5kb
EcoRV EcoRV
R primer
EcoRV
B
5’ probe
5’ probe
Wild-type allele
15 16 17
Targeted allele
Before Cre
Targeted allele
After Cre
LoxP
15 16 17*
C D
3kb
2,5kb
1kb
WT
WT/CiMKi
18 19 20
4,7kb
EcoRV
FW primer R primer
LoxP LoxP
15 16 17 18 19 20 21 22 polyA
17*
18
9,5kb
4,7kb
WT/CiMKi
WT
E
G A T
G C T
(CiMKi WT )
(CiMKi D637A )
WT
ES clone
Targeting
vector
Targeted ES
clone
Figure 2
A) CiMKi gene targeting strategy. Part of the wildtype (upper panel) and targeted (lower panel) Mps1 locus, EcoRV restriction sites,
5’ Southern probe, PCR primers and loxP sites are indicated. B) The targeted Mps1 locus following Cre recombinase addition. C)
PCR-based genotype analysis of CiMKi ES clones. Positions of PCR primers (FW/R) are indicated in A and B. D) Southern blot analysis
of CiMKi ES clones. EcoRV restriction sites and 5’ Southern probe are indicated in A and B. E) Sequences of the D637 position in the
Mps1 allele of a wildtype ES cell clone, the CiMKi D637A targeting vector and a CiMKi D637A targeted ES cell clone.
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Cre-inducible Mps1 knock-in mouse model (CiMKi)
3