title: Illumina HiSeq 2500 sequencing; Centrosome amplification is sufficient to promote spontaneous tumorigenesis in mammals
design: Centrosome amplification is sufficient to promote spontaneous tumorigenesis in mammals
library_construction_protocol: Mouse lines The Plk4Dox mouse line was created using previously described KH2 ES cells (31). KH2 ES cells possess the M2-rtTA gene targeted to the ROSA26 locus under the control of the ROSA promoter. In addition, an FRT-flanked PGK-neomycin-resistance gene followed by a promoterless, ATG-less hygromycin-resistance is targeted downstream of the Col1a1 locus to allow site-specific integration of a single copy transgene. To FLP-IN the tetracycline responsive Plk4-EYFP construct into KH2 ES cells, the mouse Plk4 ORF C-terminally tagged with EYFP was cloned downstream of the tetracycline operator and CMV minimal promoter in the pBS31 FLP-IN vector. KH2 ES cells were electroporated with pBS31-Plk4-EYFP and the pCAGGS-FLPe-puro plasmid encoding the FLP recombinase. Cells were selected with Hygromycin B and clones were amplified and checked by PCR for correct targeting. Blastocysts were injected with the targeted KH2 ES cells and chimeric mice identified. Germline transmission was detected by polymerase chain reaction analysis of tail DNA obtained at weaning. Plk4- EYFP genotyping was performed with the following primers: ACT GTC GGG CGT ACA CAA AT, CAA CCT GGT CCT CCA TGT CT and TGC TCG CAC GTA CTT CAT TC. M2-rtTA genotyping was performed with the following primers: AAA GTC GCT CTG AGT TGT TAT, GCG AAG AGT TTG TCC TCA ACC and GGA GCG GGA GAA ATG GAT ATG. Plk4-EYFP; rtTA mice were maintained by mating with C57BL6/N mice. EGFP-Centrin mice were as previously described (32). APCMin/+ mice were purchased from the Jackson Laboratory (stock 002020) and genotyped using the following primers: GCC ATC CCT TCA CGT TAG, TTC CAC TTT GGC ATA AGG C and TTC TGA GAA AGA CAG AAG TTA. Embryos and adults from both genders were included in our analysis. Mice were housed and cared for in an AAALAC-accredited facility and all animal experiments were conducted in accordance with Institute Animal Care and Use Committee approved protocols. Doxycycline Induction Mice were fed 1mg/mL doxycycline (RenYoung Pharma) in water supplemented with 25 mg/ml sucrose (Sigma). Water was changed twice per week. These Plk4-overexpressing mice developed lymphomas, squamous cell carcinomas and sarcomas between 9 and 18 months.  Genomic DNA was extracted from tissue samples using the GenElute Mammalian Genomic DNA extraction kit (Sigma) following the manufacturer’s instructions.
attributes.tag: Experimental Factor: stomach neoplasm
attributes.value: disease