Fig. 3.

Efficient recombination of Apc ^CKO in Pms2 ^cre and Lgr5-CreER intestines. (A) Laser capture microdissection-PCR was used on isolated β-gal⁺ foci to detect the recombined Apc allele (Apc ^Δ) and unrecombined Apc allele (Apc ^CKO). DNA from Pms2 ^+/+; Apc ^Δ/CKO mice, which contains equal amounts of Apc ^CKO and Apc ^Δ (1:1 control), was given an average ratio of 1 and used to normalize all experimental samples. Therefore, a ratio >1 shows a population of cells with more of the Apc ^Δ alleles, while a ratio of <1 shows a population of cells with more of the Apc ^CKO alleles. Tumors (purple diamonds) and 75% of the normal-appearing β-gal⁺ foci (blue diamonds) had a similar ratio of Apc ^Δ to Apc ^CKO, while the same β-gal⁺ foci had an increased ratio compared with either the β-gal⁻ foci (red circles) or control, heterozygous (Apc ^Δ/CKO) samples (green triangles). (B) Fluorescence-activated cell sorting was used to isolate 1000 YFP⁺ and YFP⁻ cells from crypts of either Pms2 ^cre/cre; Apc ^CKO/CKO or Pms2 ^cre/cre; Apc ^CKO/+ intestine. RNA was isolated from the different populations and then analyzed for the presence of the floxed Apc exon (exon 14). The floxed exon was lost in YFP⁺; Apc ^CKO/CKO cells, but not in YFP⁻; Apc ^CKO/CKO or YFP⁺; Apc ^CKO/+ cells. β-actin was used as a control. (C) PCR was used to detect the recombined Apc allele in intestinal sections from Lgr5-CreER; Apc ^CKO/CKO mice injected with either the high or low dose of tamoxifen. Apc was recombined in both treatments. More importantly, the 3.6-fold increase in recombined Apc DNA with high-dose treatment was in agreement with the observed 4-fold increase in β-gal⁺ crypts (see Figure 4A).