Table 1.
The effects of RG108, Scriptaid and RG108 plus scriptaid on development of porcine SCNT embryos in vitro¶.
Figure 1.
Comparison of transcription levels of twelve selected genes in blastocysts after treatment by RG108 and/or scriptaid.
Con-NT, RG-NT, Scr-NT and RG+Scr-NT indicated groups untreated, RG108 alone, scriptaid alone or by RG108 and scriptaid simultaneously, respectively. In vivo produced blastocysts (in vivo) served as the reference. Bars replaced by the “none” indicated no detected transcription. Letters of “a, b, c” on bars referred to significant (P<0.05) differences. Means labeled with the same letter or no letters didn’t differ from each other (p>0.05) and without the same letter differed significantly (p<0.05).
Figure 2.
The effects RG108 and/or scriptaid on DNA methylation levels of IGF2 DMR2 at blastocyst stage.
No obvious DNA methylation alteration in treated embryos was found after treatment by RG108 and/or scriptaid on DNA methylation.
Figure 3.
The effects of RG108 and/or scriptaid on the dynamics of DNA methylation at ICR3 of H19 during SCNT.
RG108 and scriptaid demonstrated synergetic effects on shortening the demethylation window around one division cycle with complete de-methylation at two-cell stage and re-methylation at eight-cell stage compared with the DNA methylation dynamics of Con-NT and Scr-NT embryos. At the blastocyst stage, embryos treated by RG108 and scriptaid almost rescued the semi-methylated status at H19 ICR3 compared with IVF counterparts (Figure 4 A) while not for the other two groups.
Figure 4.
DNA methylation dynamics of XIST and H19 genes in early IVF embryos.
All embryos were sex-mixed except for those at morula and blastocyst stages for XIST gene which were male.
Figure 5.
MBD3 expression was affected by RG108 and scriptaid at eight cell stage.
Treatment with RG108 and/or scriptaid can correct MBD3 levels at 8-cell stage (A) which are overexpressed in Con-NT and Scr-NT embryos compared with IVF counterparts (B).
Figure 6.
Overexpression of MBD3 at eight cell stage.
To exclude the embryos without over-expression due to failure of injection or import of plasmid DNA into the nucleus, the same molar ratio of CMV promoter-driven MBD3-coding plasmid and eGFP-coding plasmid were co-injected into the cytoplasmic of constructed oocytes, only those embryos with green fluorescence were picked(A).In addition, over-expressed MBD3 was also validated by quantitative PCR (B).
Figure 7.
Effect of over-expression of MBD3 at eight cell stage on DNA methylation levels when addition of RG108 and scriptaid.
Co-injection with eGFP-coding plasmid and pcDNA3.1+ empty plasmid with equal molar ratio (A); mock injection with the addition of RG108 and scriptaid in the culture medium (B); MBD3-injected with RG+scr (C), co-injection with MBD3-coding plasmid and eGFP-coding plasmid with medium supplemented with RG108 and scriptaid.
Figure 8.
The effects of RG108 and/or scriptaid on the dynamics of DNA methylation reprogramming of XIST during SCNT.
The 5′ upstream region of XIST gene in Con-NT embryos undertook de-methylation until at the morula stage (A), whereas a narrowed window of de-methylation occurred in both treated embryos (B, C).In addition, embryos treated by RG108 and scriptaid presented a partial de-methylation and almost fully established DNA methylation (89.2%) at blastocysts stage, which was even closer to the levels of IVF blastocysts (74.4%, Figure 4 B) compared with that of the Con-NT (33.0%) and Scr-NT (22.2%) embryos.
Figure 9.
Porcine SRY gene specific nested primers were designed to distinguish the sex of IVF embryos.
Figure 10.
The effects of RG108 and/or scriptaid on the transcription dynamics of XIST gene.
RG+Scr-NT embryos displayed a narrowed and moderate transcription activation and inactivation window similar to IVF counterparts, compared with the patterns of Con-NT and Scr-NT embryos. All embryos were sex-mixed except for those at the morula and blastocyst stages.
Table 2.
Nested primers for bisulfite-specific PCR.