Fig 1.
Schematic diagram of the mouse Sebox mRNA (NM_008759.2) and protein (NP_032785.1).
SEBOX protein has a single homeodomain (black box) near the N-terminus and is considered to be a transcription factor. N, N-terminus; C, C-terminus.
Table 1.
Primer sequences and Real-time RT-PCR conditions.
Fig 2.
Altered expression of 23 MEGs in Sebox-knockdown Mll oocytes.
Quantitative real time RT-PCR experiments were repeated at least three times, expressing data as the mean±SEM. Expression levels were calculated from the CT values after normalization with H1foo. The statistical significance was assessed by a paired t-test with p values obtained by paired t-test within the delta CT values. Asterisks, *, **, and ***, represent statistical significance at p<0.0001, p<0.01 and p<0.05, respectively. Control, GFP dsRNA-injected MII oocyte; Sebox RNAi, Sebox dsRNA-injected MII oocyte.
Fig 3.
Expression levels of selected maternal mRNAs in Sebox-knockdown 2C embryos.
To investigate the role of Sebox in degrading maternal mRNAs, Sebox RNAi knockdown PN embryos were developed to the 2C stage, and expression levels of maternal factors were determined by real time RT-PCR. Maternal factors that are typically absent in 2C control embryos, c-mos, Gbx2, Gdf9, were not degraded in Sebox-knockdown embryos arrested at the 2C stage (*p<0.05). The statistical significance was assessed by a paired t-test. Control, GFP dsRNA-injected 2C embryo; Sebox RNAi, Sebox dsRNA-injected 2C embryo.
Fig 4.
Expression of zygotic genome activation (ZGA) markers in Sebox-knockdown 2C embryos.
To investigate the role of Sebox in ZGA, Sebox RNAi knockdown PN embryos were developed to the 2C stage, and the expression levels of 13 marker genes were determined by real time RT-PCR. The expression of 4 genes (Btg1, Klf4, Kpan1 and Muerv-l) were up-regulated (A), and 4 genes (Mt1a, Rpl23, Ube2a and Wee1) were down-regulated in Sebox-knockdown 2C embryos (B); while the expression levels of 5 genes (Cdc2, Eif1a, Hsp70.1, U2afbp-rs, and Zscan4) were unchanged. Asterisks, * and **, represent statistical significance at p<0.01 and p<0.05, respectively. Control, GFP dsRNA-injected 2C embryo; Sebox RNAi, Sebox dsRNA-injected 2C embryo.
Fig 5.
Transcriptional activity assay of Sebox-knockdown 2C embryos.
Embryonic transcriptional activities were investigated by measuring embryonic nuclear EU incorporation. Control (A) and sham control (B) embryos showed normal levels of nuclear transcriptional activity, whereas transcriptional activity in Sebox-knockdown embryos (C) was diminished by comparison.
Fig 6.
Expression of Figla in Sebox-knockdown Mll oocytes.
Up-regulated expression of Figla, a known upstream regulator of SCMC, was confirmed in Sebox-knockdown Mll oocytes. Control, GFP dsRNA-injected MII oocyte; Sebox RNAi, Sebox dsRNA-injected MII oocyte.