VPA treatment increases H3K9 acetylation without affecting DNA methylation of donor cells.

<p>(A) Relative average intensity of H3K9 acetylation (H3K9ac) of control and VPA-treated cells. (B) Relative average intensity of 5-methylcytosine (5-mC) of control and VPA-treated cells. (C–D) Immunofluorescence labeling for H3K9ac of control (C) and VPA-treated cells (D). (E–F) Immunofluorescence labeling for 5-mC of control (E) and VPA-treated cells (F). The (*) denotes a significant difference between experimental groups (P<0.0001). All images were taken in the same magnification (200×).</p

Primer sequences used in real time RT-PCR.

<p>Primer sequences used in real time RT-PCR.</p

The higher levels of histone acetylation in donor cells are not maintained after nuclear transfer.

<p>(A) Relative average intensity of H3K9ac of fused couplets from control and VPA-treated groups. (B) Relative average intensity of H3K9ac of presumptive zygotes 5 h.p.a from control and VPA-treated groups. (C–D) Immunofluorescence labelling for H3K9ac of fused couplets from (C) control and (D) VPA-treated groups. (E–F) Immunofluorescence labelling for H3K9ac of presumptive zygotes 5 h.p.a. from (E) control and (F) VPA-treated groups. The asterisk (*) denotes difference between control and VPA-treated groups (P = 0.03). All images were taken in the same magnification (200×).</p

Valproic Acid increases proliferation/viability of donor cells.

<p>Proliferation/viability rates of donor cells treated with 0, 1, 2 and 5 mM VPA measured by the MTT assay. Values fitted a second grade polynomial regression (P = 0.0001).</p

Expression of <i>IGF2R</i> and <i>PPARGC1A</i> is increased in donor cells treated with VPA.

<p>The amounts of <i>IGF2R</i> (A), <i>POU5F1</i> (B) and <i>PPARGC1A</i> (C) transcripts are expressed in relation to the control group. The asterisk (*) denotes difference between control and VPA-treated groups (P<0.0001).</p

Enhanced green fluorescent protein (eGFP) expression at the fetal-maternal interface of the eGFP transgenic cloned bovine embryo at day 90.

<p>Please note (arrow) that the trophoblastic cells that are close to the maternal cells displays weak staining for eGFP. Fetal mesenchyme (FM); endometrial epithelium (EE); maternal stroma (MS) and villous area (VA).</p

Detection of eGFP protein in fetal and maternal tissues from eGFP transgenic cloned placenta.

<p>Western blotting analysis to detect eGFP protein in 1: transgenic eGFP fetal chorion; 2: transgenic eGFP placentome; 3: molecular marker and 4: intercaruncular endometrium from pregnant horn eGFP from transgenic placenta.</p

Primer/Probe sets used for qRT-PCR.

a<p>Forward =  sense (5′) primer.</p>b<p>Each probe was synthesized 6-FAM reporter dye and TAMRA quencher.</p>c<p>Reverse =  antisense (3′) primer.</p

Expression of eGFP in fetal and maternal tissues from eGFP transgenic cloned placenta.

<p>Mean of relative expression of eGFP mRNA in the intercaruncular chorion (black bar), placentome (dark grey bar) and intercaruncular endometrium (light grey bar).</p

Expression of eGFP protein in the placentome at day 90.

<p>(A) On top the fetal side (FS) containing arcade zone (AZ) and villous areas (VA) with close intermingling of fetal and maternal tissues (MT). (B–C) Details of the arcade zone showing the trophoblast cells (T) and maternal uterine epithelium (M) positive for eGFP; endometrial stroma (EE) was mostly negative. Details of the arcade zone showed similar staining, including a binucleated trophoblast giant cell (arrows). Please note that some binucleate trophoblast giant cells are strong positive for eGFP while others are not. In (D–F), the presence of syncytial regions (arrows) in the villous region. Please note the fetal cells in close contact to the maternal epithelium displays weak staining to eGFP (arrows). (G) Negative control: placenta derived from a normal pregnancy that was stained for eGFP detection. trophoblast (T), endometrial stroma (EE), maternal uterine epithelium (M).</p