A senescence-inflammatory switch from cancer-inhibitory to cancer-promoting mechanism
A senescence-inflammatory switch from cancer-inhibitory to cancer-promoting mechanism
For generation of conditional CKI knockout mice, Cre was transiently expressed in ES cells to produce specific deletion of the neomycin cassette and an intact floxed CKI alpha allele. A second aggregation was done, and Vilin-Cre-ERT2 transgenic mice were used as a gut-specific deleter to generate a conditional CKI floxed/floxed Villin-Cre-ERT2 knockout mouse (CKI alpha KO). Inducible double-knockout mice (p53 and CKI alpha) were generated by crossing mice in which CKI alpha was floxed with mice in which p53 was floxed (CKI alpha/p53 DKO). Mice were kept under specific pathogen-free conditions at the Hadassah Medical School of the Hebrew University. Mice were injected with 150 milligram/kilogram Tamoxifen (Sigma) subcutaneously, at a regimen of 6 injections every other day. Sulindac (Sigma) was administered in the drinking water at 0.6 gram/liter, one week before the first Tamoxifen injection, and throughout the 2 weeks of knockout induction, for a total period of 3 weeks. BV6 (Genentech) was injected intraperitoneally at 10 milligram/kilogram, every other day, from day 2 of knockout induction. BOT64 (Santa Cruz) was injected intraperitoneally at 30 milligram/kilogram, every day, from day 2 of knockout induction. 1-2 days after the last Tamoxifen injection, mice were injected intraperitoneally with 10 microliter/gram of 5-bromo-2-deoxyuridine (BrdU; GE Healthcare) and sacrificed 2 hours later, on day 10-12 of knockout induction. Jejunum, ileum and the entire large intestine were flushed with ice-cold PBS, cut open longitudinally, and subjected to fixation in 4% formaldehyde and paraffin embedding(FFPE). Intestinal epithelial cells (IECs) were isolated from the middle part of the small intestine and separated into single cells in HBSS containing 10 millimolar  HEPES, 5 millimolar EDTA and 0.5 mM DTT, at 37C for 30 minutes. Total RNA was extracted from IECs using TRI-reagent (Sigma) and phenol/chloroform methods. Total RNA from IECs of three mice per genotype was pooled, processed with the mRNA Sequencing Kit (Illumina). Library construction was done with the Illumina TRUSeq kit according maufacturers instructions.
Experimental Factor: genomic modification
flox sites around one CKI alpha allele
Experimental Factor: genotype
cki alpha -/+