Fig 1.
Parthenogenetic blastocysts and zona pellucida (ZP).
After electrical activation, the embryos were cultured for 7 to 8 days. Blastocysts and freed ZP (asterisks) with various sizes and shapes were observed. (A) The thin, stretched, ruptured, empty zona were observed after shedding. (B) The empty zona with a large slit (solid arrowhead) or with a narrow slit (open arrowhead) were discarded. Only empty zona shaped like the video game icon "Pac-Man" with a proper slit size were collected. (B`) Bar in B`is 100 μm.
Fig 2.
Injection of 4-cell stage embryos into empty zona.
After dissolving the zona pellucida (ZP), the denuded parthenotes at the 4-cell stage (A) were injected into the empty zona via micromanipulation with hollow glass needles (B-G). Multiple embryos with various numbers were reconstructed using the empty zona method (H and I). The embryos injected into an empty zonae were gently pressed around through the glass needle, causing them to bind together more tightly (J-L).
Fig 3.
Representative images of differentially-stained porcine day 6 blastocysts.
A singleton (A) and 3X aggregate blastocyst with compact (B) or scattered or non-compact (C) inner cell mass (ICM). Using a conventional differential staining method, ICM were labeled with Hoechst 33342 (blue) and trophectoderm (TE) nuclei with propidium iodide (red); photographed under an epifluorescent microscope. Scale bars: 100 μm.
Table 1.
Developmental rate of triple-aggregated embryos produced using the EZ and conventional microwell methods.
Fig 4.
Aggregate formation and in vitro development of embryos using the empty zona method.
Chimeric morulae and blastocysts derived from 2 (2X), 3 (3X), 4 (4X), 5 (5X), 6 (6X), 7 (7X) and 8 (8X) aggregated embryos were photographed using an optical microscope. The embryos began to compact on day 2 of culturing after introduction and the majority developed to the blastocyst stage.
Table 2.
Aggregate formation and developmental potentials of aggregates with various embryo numbers using the empty zona method.
Table 3.
Aggregate formation and in vitro development of cloned embryos expressing GFP and RFP.
Fig 5.
Green fluorescent protein (GFP) and red fluorescent protein (RFP) expression in 4X cloned aggregates using the empty zona method.
Two cloned embryos derived from the GFP or RFP cell line were aggregated using the empty zona method. A cloned aggregate (4X) was photographed (A) and the cell distribution pattern traced using a fluorescent microscope.