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Adenovirus-Cre-mediated recombination in mammary epithelial early progenitor cells

Baylor College of Medicine, Department of Molecular and Cellular Biology, One Baylor Plaza, Houston, TX 77030, USA

View larger version (23K): [in a new window]   Fig. 1. AdCre1-mediated mammary gland epithelium-specific gene modification. Mammary gland tissue was harvested from all ten glands of late pregnant (14-18 days) animals bearing a floxed allele. Mammary gland tissue was dissociated and enriched for epithelial cells. Those cells were plated on serum/fetuin-coated plastic at a cell density of 2.5x105/cm2 and cultured for 3 days. Cells were infected with AdCre1 at a MOI 50 and cultured for 2 more days. Cells were injected in epithelium-free fat pads of a 3-week-old host animal on day 5 of culture and allowed to grow out for more than 7 weeks.

View larger version (39K): [in a new window]   Fig. 2. Reporter construct and recombination analysis by PCR. (A) Map of the wild-type (WT) and R26R alleles, and the recombined R26R allele (Soriano, 1999). The LacZ gene was expressed upon Cre-mediated recombination. Primers used for PCR detection of genotype were 1/3 (WT), 1/2 (R26R), detection of recombination 1/Z3 (600 bp fragment) and detection of LacZ Z1/Z2 (Araki et al., 1995). (B) PCR analysis for Cre-mediated recombination of the R26R allele in (1) mammary gland epithelium in culture (P): -AdCre1: panel I, lanes 7,8, and panel II, lanes 1,10; +AdCre1: panel I, lanes 9,10 and panel II, lanes 2,9; (2) mammary epithelium outgrowth (O): -AdCre1: panel I, lanes 3,4 and panel II, lanes 5,6; +AdCre1: panel I, lanes 1,2 and panel II, lanes 3,4,7,8; (3) WT mouse tail DNA (M-): panel I, lane 11 and panel II, lane 11; (4) non-DNA control (-DNA): panel I, lane 12 and panel II, lane 12; (5) marker, 50 bp DNA ladder: panel I, lane 13; (6) WT mammary gland DNA (#3C): panel I, lanes 5,6.

View larger version (139K): [in a new window]   Fig. 3. ß-Galactosidase activity detected in mammary gland epithelial cells in primary cultures and AdCre1-infected mammary gland epithelial outgrowths. (A) 80% of MECs infected with Adß-gal at an MOI of 50 are ß-galactosidase positive, magnification 100x; (B) not infected with Adß-gal, 100x. (C) 71% of R26R MECs infected with AdCre1 at an MOI of 50 are ß-galactosidase positive, 100x. (D,E) X-gal and whole mount staining with carmine aluminum of two primary outgrowths of a single primary culture 15 weeks post transplantation. 9472R, 15 day pregnant, 40x (D); 9471R, virgin, 10x (E).

View larger version (125K): [in a new window]   Fig. 4. X-gal staining in secondary transplants of AdCre1-infected mammary epithelium. (A) 2443L, virgin, magnification 20x; (B) 2442L, 12.5 day pregnant, 20x; (F) 2448R, 1 day lactation, 20x; (C) 2448R, 10x, X-gal and carmine whole mount staining. Hematoxylin- and eosin-stained sections of outgrowths are shown in A-C. (D) 2443L, 100x; (G) 400x, 2442L; (E) 100x; (H) 400x; (I) 2448R, 400x.

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