FIG. 1.

Proviral MMTV content in normal mammary glands and tumors. Viral strain-specific PCR products (BALB/2, BALB/14, and LA; primers shown in Table 1) were obtained from DNA of the following: (A) MMTV-uninfected normal mammary glands (MG), MMTV-infected mammary glands from virgin females (V), females after third pregnancy (3P) or after seventh pregnancy (7P), and primary mammary tumors (T1); (B) pregnancy-independent passages from eight different tumor lines. For A and B, each bar graphic and ethidium bromide-stained gel shows a representative assay from at least three independent experiments. PCR conditions: 94°C 2 min, followed by 27 to 30 cycles of 94°C for 30 s, 55 to 58°C for 30 s, and 72°C for 1 min, followed by 72°C for 5 min. In panel A, bar graphics show qPCR relative product quantification normalized to an endogenous host sequence (e) (accession no. AL833773.6: 11076-5′-GGGTGTTCTTGATCCATTTGG-3′-11058 and 11253-5′-CACACAGCTATGGTCACTTG-3′-11273) using 1:30,000 SYBR Green (Molecular Probes, Inc). Proviral MMTV/e, proviral MMTV content relative to endogenous host sequence; a value of 1 was assigned to the sample showing the highest level of proviral MMTV content; au, arbitrary units. In panel B, Southern blot analysis with an MMTV-LTR probe (3, 9) is shown to indicate fragments that have been cloned out by IPCR (big arrows); arrowheads, other MMTV insertion sites that have not been isolated; PD, pregnancy-dependent tumor; PI, pregnancy-independent tumor. Top right corner: MMTV-LTR probe Southern blot analysis showing the endogenous MMTV band pattern from BALB/c mouse spleen DNA digested with EcoRI and HindIII.