Full-term development of embryos cultured in the warm box.

<p>Key: Gas perm. film, gas-permeable film; M/B, Morula/Blastocyst; Frag, fragmentation; implant., implantation</p><p>Full-term development of embryos cultured in the warm box.</p

Abnormalities in placentae derived from cloned embryos.

<p>(A) Placental weights from ICSI-derived control embryos and CB- or LatA-treated cloned embryos. Error bars indicate SD. Asterisks indicate significant difference at p < 0.05. (B) Hematoxylin and eosin staining of placentae derived from ICSI-derived control and CB- or LatA-treated cloned embryos. Abnormal distortion of the boundary between the spongiotrophoblast and labyrinth layers was observed in placentas derived from both CB- and LatA-treated cloned embryos. Bar = 500 μm.</p

Enucleation of MII chromosomes from oocytes with fluorescence observation using halogen lamp.

<p>The MII chromosomes of bovine oocytes are normally invisible by conventional microscopy because of dark lipid droplets in the ooplasm (A). After labeling with an H3S10ph antibody–phycoerythrin conjugate, the MII chromosomes could be recognized clearly and this allowed us to remove them along with minimal cytoplasm (B). MII chromosomes in porcine oocytes were detected by the same method (C). Some chromosomes (arrow) were left occasionally inside the cytoplasm of mouse oocytes after enucleation (D). Live and healthy cloned mice (brown) were obtained after enucleation using this method without any significant decrease in the success rate (E). Enucleation of the MII spindle from mouse oocytes with this system. (F) Before, (G) during and (H) after enucleation.</p

Production of cloned mice using phycoerythrin and antibody injected oocyte.

<p>Production of cloned mice using phycoerythrin and antibody injected oocyte.</p

The newly designed filter adapter and its applications.

<p>The adapter is placed on the top of the condenser (A). This adapter has a diaphragm (B) and a gap between the filter and frame (C). (D) Bright field and (E) fluorescence of green fluorescent protein (GFP)-labeled embryonic stem (ES) cells in the ICM of a chimeric mouse blastocyst. When the diaphragm is opened, halogen light can pass around the filter and a merged image can be obtained (F). This filter adapter could also be useful in purifying rare cell populations. Typically, fluorescence-activated cell sorting is used for this purpose, but cannot be applied if samples do not contain sufficient cell numbers. We collected spermatogonia from neonatal mouse testes using a CD9 antibody labeled with phycoerythrin. The CD9-positive spermatogonia could be discriminated from other cells and we were able to collect them using a micromanipulator without the need for a mercury vapor lamp. (G) Spermatogonia were detected using a CD9 antibody–phycoerythrin conjugate and positively stained cells (arrows) were drawn into a micropipette using a micromanipulator (H).</p

Full-term development of embryos cultured in microtubes with or without a gas-permeable film.

<p>Values with different superscript letters are significantly different (<i>P</i> < 0.05 by χ² tests).</p><p>Key: Gas perm. film, gas-permeable film; M/B, Morula/Blastocyst; Frag, fragmentation; implant., implantation</p><p>Full-term development of embryos cultured in microtubes with or without a gas-permeable film.</p

Microtubes and the simple warm box.

<p>(A) Puncturing a microtube lid using scissors. (B) A hole in the microtube lid facilitates gas exchange. (C) A microtube capped with a gas-permeable film. (D) Tubes were transferred into a plastic bag with the special gas mix. (E) Side view of the warm box showing a temperature indicator in the center. (F) A plastic bag placed in the box.</p

Histone modifications in 1-cell stage embryos.

<p>(A-D) acH3K9, acH3K14, H3K9me2 or H3K4me2 levels in ICSI-generated and CB- or LatA-treated cloned embryos. Bar = 30 µm. (E) The intensities of immunofluorescence for acH3K9, acH3K14, H3K9me2 and H3K4me2 relative to that of H2B. They are compared with the intensities in ICSI-generated control embryos. The acetylation or methylation levels of these regions were not different between LatA- and CB-treated cloned embryos.</p

Staining of MII spindle using phycoerythrin and observation.

<p>The antibody–phycoerythrin conjugate diffused immediately after microinjection into mouse oocytes (A) but the conjugate could bind to the MII chromosome array within a few minutes (B, C). Five minutes later, the chromosomes became sufficiently bright for enucleation (D). The brightness of MII chromosomes was measured by comparison with the cytoplasm; the luminance increased with time up to 8 min (E). Imaging could reveal individual chromosomes (F, G). Different concentrations of the phycoerythrin conjugate were microinjected into oocytes (H–K). The staining intensity was proportional to the concentration of conjugate used and 75 µg/mL of antibody was the minimum needed for clear observation. Fading of the phycoerythrin label was examined (L–O). (L, M) Images of phycoerythrin-injected oocytes observed using a conventional fluorescence microscope faded within 30 s. (M, N) When these samples were observed using the halogen light system, the image did not fade even when observed continuously for over 10 min.</p

Full-term development of inbred or outbred mouse strain embryos transported by parcel-delivery service.

<p>Key: Gas perm. film, gas-permeable film; M/B, Morula/Blastocyst; Frag, fragmentation; implant., implantation</p><p>Full-term development of inbred or outbred mouse strain embryos transported by parcel-delivery service.</p