Fig. 2. Generation and characterization of Zp3-Foxo3a Tg mice. (A) DNA construct for generation of the Tg mice. The construct consists of a 6.5 kb Zp3 promoter, a 2 kb constitutively active Foxo3a cDNA (Foxo3a-TM) with a FLAG tag, and a 0.3 kb bovine growth hormone (BGH) poly-A tail. P1 and P2 indicate the positions from which the two genotyping PCR primers were derived. I-SceI, SmaI and NotI restriction sites are indicated. (B,C) The expression of constitutively active Foxo3a-FLAG in ovaries of Tg mice was confirmed by RT-PCR where a band of approximately 1.3 kb was detected (B), and by detection of the FLAG tag at the C-terminus of the exogenous Foxo3a protein in ovarian lysates of Tg mice (C). Ovaries from 15- to 17-day-old Zp3-Foxo3a Tg mice and WT littermates were homogenized and subjected to western blotting for FLAG tag and ß-actin expression. (D,E) Localization of Foxo3a antigen in ovaries of 8-day-old Tg mice and WT littermates. Immunohistochemistry confirmed Foxo3a expression in the nuclei and cytoplasm of oocytes from primary follicles (D, arrow) and secondary follicles (not shown) of Tg mice, but not in oocytes from similar follicles in WT ovaries (E, arrows). Nuclear expression of Foxo3a in oocytes of primordial follicles, which is believed to be endogenous expression, was also detected in both Tg mice and WT mice (D,E, arrowheads). The experiments were repeated three times and representative results are shown.