Generation of ApcCKO ES cells and mice.
The linearized targeting vectors were electroporated into 129/S-derived ES cells, WW6 cells [15]. All candidate G418-resistant ES clones were screened by long-range gene-specific PCR using Expand Long Template PCR System (Roche, Indianapolis, Indiana, United States), followed by sequencing to validate the correct insertion of the single loxP site and the selection cassette. Two ES clones with correct ApcCKON/+ modification were injected into C57BL/6J blastocysts, after which chimeric mice with high levels of ES cell contribution were backcrossed to C57BL/6J females to produce heterozygous F1 offspring. The genomic DNA samples obtained from the tail of F1 offspring were subsequently analyzed by Southern blot analysis to further confirm the correct homologous recombination. By crossing the heterozygous mice to FLPe deleter mice [34], PGKneor cassette was deleted in the germline by FRT-mediated recombination to generate mice with the final ApcCKO allele. ApcCKO heterozygous mice were crossed together to generate homozygous mice, and the homozygous offspring were interbred for maintenance.