Fig. 1. Conditional deletion of Pten in mammary gland. (A) LoxP sequences are inserted on both sides of exon 5. After crossing with MMTV-Cre^+/– transgenic mice, exon 5 is deleted in the mammary gland. P1, P2 and P3 localize the PCR primers used for genotyping. The predicted PCR products are: loxp=1100 bp; wild type=1000 bp; 5=300 bp. (B) PCR screen for Cre-mediated Pten exon5 deletion in Pten^loxp/loxp/MMTV-Cre^+/– mammary glands at different developmental stages. Lanes 1-4, control DNAs isolated from wild type (WT), Pten^loxp/+, Pten^loxp/loxp and Pten^loxp/5 mice; lane 5-8, DNAs prepared from 5 week virgin, 8 week virgin, 3 days after weaning, and resting multi-parous mammary glands. (C) Decreased PTEN protein expression in the Pten^loxp/loxp/MMTV-Cre^+/– mammary gland. Total cell lysates from mutant (Pten^loxp/loxp/MMTV-Cre^+/–, M) and control (Pten^loxp/loxp/MMTV-Cre^–/–, C) mice of 6 week-old virgin (V-6wk), pregnancy day 13 (P13), lactation day 10 (L10), and post-weaning day 1 (PW1) were subjected to western blot analysis, using -PTEN antibody and -actin antibody for loading controls. (D) Deletion of PTEN in mammary epithelial cells. Immunohistochemical staining for PTEN is conducted on mutant and control mammary glands at day 13 of pregnancy. Scale bar: 20 µm.