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. 2013 Jul 29;210(8):1529–1544. doi: 10.1084/jem.20121337

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© 2013 Gorrini et al.

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Figure 4. — Brca1-deficient pMECs can regenerate a functional mammary gland but have a limited lifespan in vivo. (A) Breeding strategy used to obtain B1^f/fLuc^f/+, KLuc^f/+, and KB1^f/fLuc^f/+ animals used for in vivo fat pad transplantation assays. pMECs isolated from B1^f/fLuc^f/+, KLuc^f/+, and KB1^f/fLuc^f/+ donor mice were transplanted in 21-wk-old recipient mice to generate outgrowths. (B) In vivo Luc activity in outgrowths derived from B1^f/fLuc^f/+ (mouse 1 as negative control), KLuc^f/+ (mice 2 and 8 as positive controls), and KB1^f/fLuc^f/+ (mice 3–7) pMECs at 4 and 24 wk after transplantation. (C) Quantitation of Luc activity shown in B using Living Image 3.0 software. (D) qPCR of Brca1 WT allele with genomic DNA from pMECs isolated from KLuc^f/+ and KB1^f/fLuc^f/+ 4-wk outgrowths. (E) Immunohistochemical staining of 4-wk KLuc^f/+ and KB1^f/fLuc^f/+ outgrowths with anti-Luc and anti-CK14 Abs. Phase-contrast images of the same glands are shown below. (F) Whole-mount staining of KLuc^f/+ and KB1^f/fLuc^f/+ 4-wk outgrowths. Terminal end buds are shown at higher magnification. (G) Hematoxylin-eosin staining of KLuc^f/+ and KB1^f/fLuc^f/+ outgrowths at 24 wk after transplantation. Data are representative of 10 outgrowths examined per genotype. Bars: (E) 16 µm; (F and G) 50 µm. (H and I) RT-PCR analysis of p16/INK4 (H) and p19/ARF (I) mRNA levels in KLuc^f/+ and KB1^f/fLuc^f/+ 8-wk outgrowths. (J) pMECs from KLuc^f/+ and KB1^f/fLuc^f/+ 8-wk outgrowths were stained with FITC-γH2AX and analyzed by flow cytometry. (K) p21/Cdkn1a mRNA levels in pMECs from KLuc^f/+ and KB1^f/fLuc^f/+ 8-wk outgrowths. (C, D, and H–K) Data represent the mean ± SEM of n = 5 outgrowths of each genotype. *, P < 0.05; **, P < 0.01.