Figure 1.
Shp1 fl/+;MMTV-Cre and MMTV-Cre display a lactation defect.
Shp1 fl/+ (control), Shp1 fl/+;MMTV-Cre, and MMTV-Cre female mice were mated with wild type FVB male mouse. On the first day following parturition, the litter size was normalized to 6 pups/litter. Pups were weighted at lactation days 5, 8, and 10. The average weight of each litter is shown and bars indicate the standard deviation (SD). ** indicates P<0.001. The data shown is representative of at least two independent experiments.
Figure 2.
Impaired differentiation and precocious involution of mammary epithelial cells in mice expressing the MMTV-Cre transgene.
(A) The number 4 mammary glands from Shp1 fl/+ (control), Shp1 fl/+/;MMTV-Cre, and MMTV-Cre mice were isolated from 10-week old virgin mice and subjected to whole mount analysis as described in the Materials and Methods. Scale bar = 5 mm. (B, C) Six-week old Shp1 fl/+ (control), Shp1 fl/+;MMTV-Cre, and MMTV-Cre female mice were mated with male wild type mice. The number 4 mammary glands were removed at pregnancy day 15 (P15), lactation day 1 (L1), and day 10 (L10), and processed for histological analysis as described in the Materials and Methods. Images were taken under lower (B) and higher (C) magnification respectively. Scale bars represent 100 µm in (B) and 50 µm in (C). Similar results were seen from at least two different mice for each genotype. (D, E) Analysis of apoptotic cells in L10 mammary glands as described in (B, C). (D) Sections of L10 mammary glands from indicated mice were stained with antibody against cleaved caspase 3 (see Materials and Methods). Cells positive for cleaved caspase 3 are stained dark-brown. Images were taken using 40X objective lens. Scale bar = 50 µm. (E) Quantification of caspase 3 positive cells from (D) (see Materials and Methods). Two mice from each genotype were analyzed. **p<0.01 and ***p<0.001.
Figure 3.
Changes in the activation of Stat5 and Stat3 in mammary glands from mice expressing the MMTV-Cre transgene during late pregnancy and early lactation.
The number 4 mammary glands from Shp1 fl/+ (control) (lane 1), Shp1 fl/+;MMTV-Cre (lane 2), and MMTV-Cre (lane 3) mice at pregnancy day 15 (P15), lactation day 1 (L1), day 10 (L10), and from wild type FVB mice at involution day 2 (I2) (lane 4) were isolated and whole tissue lysates prepared as described in the Materials and Methods. Equal amount of proteins (∼40 µg) from each sample were resolved by gel electrophoresis, and immunoblotted with antibodies against phospho-Stat5 (p-Stat5; Y694), or phospho-Stat3 (p-Stat3; Y705). The immunoblots were reprobed with antibodies against Stat5, Stat3, and cytokeratin-18 (CK-18), and beta-actin to demonstrate equal loading. Similar results were seen from at least two different mice for each genotype.
Figure 4.
Identification of the linkage of the K14-Agouti cassette to the MMTV-Cre transgene using inverse PCR.
(A) The schematic diagram of the inverse PCR. (B) PCR using specific primer sets identified the K14-agouti cassette adjacent to the MMTV-Cre transgene. The linkage of the linkage of the MMTV-Cre and K14-agouti transgenes allow distinction of the line A, line D and line F founder lines. The MMTV-Cre cassette [5] also contains a rabbit growth hormone intron sequence (RGI). The K14-Agouti cassette [14] also contains a human growth hormone polyadenylation sequence (GHA). The Cre-F/Cre-R primer set identifies the presence of Cre gene. The K14P-3R/GHA-1F primer set identifies the presence of the K14-agouti cassette. The MMTV-1R/GHA-1R primer set identifies the presence of the head-to-tail linkage of the K14-agouti cassette and the MMTV-Cre cassette. The RGI-1F/GHA-1R primer set identifies the presence of the tail-to-tail linkage of the MMTV-Cre cassette and the K14-agouti cassette. 1, wild type DNA; 2, MMTV-Cre DNA used in this study; 3, MMTV-Cre line A DNA from Jackson lab; 4, MMTV-Cre line D from Jackson lab; 5, MMTV-Cre line A DNA from Hennighausen lab; 6, MMTV-Cre line F DNA from Hennighausen lab; N, no DNA control. Note, lane 2, 3, and 5 (all Line A) have the same PCR fragments for the head-tail and the tail-tail linkages whereas lane 4 (line D), and lane 6 (line F) only have PCR fragments for the tail-tail linkage. The sizes of PCR products are listed under each DNA gel panel.
Table 1.
Primers used for inverse PCR and genotyping the MMTV-Cre mice.