Additional file 1 of Evaluating histone modification analysis of individual preimplantation embryos

Additional file 1

Effects of si<i>Ing3</i> injection on asymmetric cell division in mouse oocytes.

<p>(A) <i>Ing3</i> mRNA was significantly decreased in the fully grown GV oocytes injected with si<i>Ing3</i> (*<i>p</i><0.05). (B) ING3 protein levels, after normalization to α-tubulin, were decreased in fully grown GV oocytes injected with si<i>Ing3</i>. (C) Abnormal cell division was observed in several si<i>Ing3</i>-injected oocytes at the MII stage (arrows). (D) In si<i>Ing3</i>-injected oocytes, the rate of symmetric division was significantly increased as compared to that observed in siControl-injected oocytes (*<i>p</i><0.05). (E) Maturation rates were not different between si<i>Ing3</i>- and siControl-injected oocytes.</p

Effects of si<i>Ing3</i> injection on the levels of AcH4K12, asymmetric cell division–related gene expression, and mTOR protein in mouse oocytes.

<p>(A) The levels of AcH4K12 in fully grown GV oocytes cultured for 15.5 h after injection of si<i>Ing3</i> were significantly decreased as compared to controls. Red, AcH4K12; blue, chromatin. (B) <i>mTOR</i> mRNA levels were significantly decreased after si<i>Ing3</i> injection in fully grown GV oocytes (*<i>p</i><0.05) as compared with other asymmetric cell division–related genes. (C) Although the protein localization of mTOR was not changed, the amount of mTOR protein was reduced in si<i>Ing3</i>-injected oocytes. Green, mTOR; blue, chromatin. (D) The expression levels of mTOR-downstream genes, such as <i>Cdc42</i>, <i>Rac1</i>, and <i>RhoA,</i> in fully grown GV oocytes were significantly decreased 15.5 h after si<i>Ing3</i> injection (*<i>p</i><0.05).</p

Effects of si<i>Ing3</i> injection on cortical reorganization in mouse oocytes.

<p>(A) Actin cap formation was noted in siControl-injected oocytes at the MI, ATI, and MII stages. By contrast, no actin cap was formed in si<i>Ing3</i>-injected oocytes at any stage. Arrows indicate the actin cap. Green, α-tubulin; red, actin; blue, chromatin. (B) CGs were absent in the cortex where the chromosomes were located at the MI and MII stages in siControl-injected oocytes. By contrast, in si<i>Ing3</i>-injected oocytes, CGs were distributed throughout the cortex at the MI and MII stages, and were intensely localized in the region of cell adhesion at the MII stage. Circles and arrows denote the CGFD. Green, cortical granules; blue, chromatin.</p

Localization of ING3 during mouse oocyte maturation.

<p>(A) ING3 was predominantly localized in the nucleus in fully grown GV oocytes. After GVBD, ING3 localized homogeneously throughout the cytoplasm. Green, ING3; blue, chromatin. (B) ING3 bound to the chromatin in fully grown GV oocytes. Green, ING3; blue, chromatin.</p

Aggregation of pnbiPSCs into <i>in vitro</i> fertilized embryos, and their development <i>in vitro</i>.

<p>*RFP vector was introduced</p><p>Aggregation of pnbiPSCs into <i>in vitro</i> fertilized embryos, and their development <i>in vitro</i>.</p

Phase-contrast images of biPSCs established in two different culture conditions.

<p>(A) bADCs. (B) Primary colonies appearing in primed cell-culture medium. (C) Established primed-type biPSCs. (D) Colonies converted from the primed to naïve state. (E) Primary colonies appearing in niPSCs medium. (F) Established naïve-type biPSCs. (A)–(C), (E), scale bars = 500 μm. (D), (F), scale bars = 100 μm.</p

Production of chimeric fetuses from bovine embryos using the aggregation method.

<p>(A) Naïve-type biPSCs expressing Tag-RFP were aggregated with host at the 8- to 16-cell stage of <i>in vitro</i> fertilized embryos. (B) Chimeric fetuses at day 90 of gestation derived from aggregated embryos. (C) PCR analysis using transgene-specific primers for genomically integrated Oct3/4-2A-Klf4 sequences in 14 tissues. Genomic DNA isolated from pnbiPSCs was used as a positive control. H₂O was used as a negative control (buffer alone for RT-PCR). Immunofluorescence analysis showing the distribution of pnbiPSC-derived cells (RFP-positive with red signals) in the small intestine (D), placenta (E), gonad (F, VASA-positive cells with green signals; arrowheads indicate the portion that is double-positive for RFP and VASA), and kidney (G) of the chimeric fetus. Nuclei were stained with DAPI (blue).</p

Differentiation potential of biPSCs in culture.

<p>(A) Embryoid body formation of pbiPSCs grown for 6 days in low cell-adhesion dishes. Immunocytochemical staining for markers for the three germ-layer in differentiated cells derived from pbiPSCs. α-fetoprotein (B, endoderm), actin smooth muscle (C, mesoderm), and glial fibrillary acidic protein (D, ectoderm) were used as markers. (E) Embryoid body formation by pnbiPSCs. Immunocytochemical staining for α-fetoprotein (F), actin smooth muscle (G), glial fibrillary acidic protein (H). (A), (E), scale bars = 500 μm. (B)–(D), (F)–(H), scale bars = 100 μm.</p

Reprogramming of bovine amnion-derived cells (bADCs) into iPSCs using Dox-inducible PB vectors.

<p>(A) Timeline for the establishment of primed-type biPSC lines. (B) Timeline for the establishment of naïve-type biPSC lines.</p