ERBB4 transgene expression. (A) Plasmid map for MMTV-ERBB4 carrying SalI and AatII sites. SalI-AatII double digestion of the plasmid separated the ERBB4 CYT-1(A1) and CYT-2 (A2) inserts, which were gel purified for micro-injections to generate CYT-1 and CYT-2 ERBB4 transgenic mice. (B) ERBB4 mRNA expression in the mammary glands of CYT-1 and CYT-2 mice at various developmental stages. RNA was extracted from mammary glands from three mice at each stage and pooled. Relative ERBB4 expression levels are represented by vertical bars as measured by real-time quantitative reverse transcription PCR using Taqman primers, with error bars indicating the standard deviation for samples run in triplicate. (C) ERBB4 immunohistochemical staining (detecting both mouse endogenous and human transgenic ERBB4) in nonpregnant multiparous female transgenic and control FVB mice. β2m, mouse-specific beta-2 microglobulin; MMTV, mouse mammary tumor virus.