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Volume 3, Issue 4, Pages 557-564 (April 2001) Direct Exposure of Mouse Ovaries and Oocytes to High Doses of an Adenovirus Gene Therapy Vector Fails to Lead to Germ Cell Transduction Jon W. Gordon Molecular Therapy Volume 3, Issue 4, Pages 557-564 (April 2001) DOI: 10.1006/mthe.2001.0290 Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 1 LacZ staining of ovaries 5–7 days after injection of Ad0-gal directly into the ovary (A) or under the ovarian bursa (B). Note intense LacZ staining, with follicles and oocytes (arrows) completely negative (original magnification ×100). C and D are negative controls. Molecular Therapy 2001 3, 557-564DOI: (10.1006/mthe.2001.0290) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 2 Immunohistochemical localization of LacZ protein on paraffin embedded sections. LacZ activity is clearly present in the interstitial tissue as black deposits, while a clearly visible follicle with an oocyte (arrow) is entirely negative. Molecular Therapy 2001 3, 557-564DOI: (10.1006/mthe.2001.0290) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 3 A large group of 2- to 4-celled embryos fertilized in vivo after ovarian injection of Adβ-gal and stained in vitro for LacZ enzymatic activity. (A) These embryos at 4× original magnification and (B) at 10× original magnification are shown. Note that despite intense staining of positive control embryos, the experimental embryos are completely negative for LacZ staining. Molecular Therapy 2001 3, 557-564DOI: (10.1006/mthe.2001.0290) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 4 A group of morulae stained for LacZ after exposure of unfertilized eggs to Adβ-gal, washing, and insemination. Three embryos exposed to the same titer of the vector at the two-cell stage for 1 h were stained simultaneously and are shown as a separate small group at the upper right. Note intense staining of positive control embryos with completely negative staining of embryos derived from exposed oocytes. Molecular Therapy 2001 3, 557-564DOI: (10.1006/mthe.2001.0290) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 5 Embryos stained for LacZ after oocytes were exposed to the vector and washed once (A), twice (B), or three times (C). Progressive diminution of staining with repeated washing is apparent. In A (one wash), several blastomeres of two embryos are intensely stained. In B (two washes), two blastomeres of one embryo show moderate staining (arrow), and in C (three washes), very faint staining of a single blastomere is observed (arrow). For these experiments the vector containing the RSV promoter (11) was used. Molecular Therapy 2001 3, 557-564DOI: (10.1006/mthe.2001.0290) Copyright © 2001 American Society for Gene Therapy Terms and Conditions