To further promote the early development of porcine embryos and capture "naïve" pluripotent state within blastocyst, the experiment explored the effects of lysophosphatidic acid (LPA) on the early development of porcine parthenogenetic embryos and the expression of pluripotency relevant genes. The results showed that the addition of 50 μM LPA significantly improved parthenogenetic embryo cleavage rate (82.7% vs. 74.7%, p < 0.05), blastocyst rate (24.5% vs. 11.3%, p < 0.05) and blastocyst cell count (56 ± 7.9 vs. 42 ± 1.0, p < 0.05) than that of the control group. In addition, immunostaining experiment determined that the fluorescence intensity of OCT4 was also significantly higher than that of the control group. The quantitative real-time polymerase chain reaction (qRT-PCR) test revealed that addition of 50 μM LPA could significantly enhance the expression level of pluripotent gene OCT4 and trophoblast marker genes CDX2, however, decrease the expression of primitive hypoblast marker gene GATA4. The results also indicated that LPA might decrease the expression of GATA4 through the ROCK signalling pathway. For further investigating the effect of the addition of LPA on the expression of "primed" and "naïve" genes, we also detected the expression of those pluripotency-related genes by qRT-PCR. The results showed addition of LPA had no significant effect on the expression of "naïve" pluripotent genes, but it was able to significantly decrease the expression of "primed" pluripotent genes, NODAL and Activin-A; furthermore, it also could significantly improve the expression of OCT4 and c-Myc which act as two important ES cell renewal factors. Above all, the addition of LPA can facilitate the early development of porcine parthenogenetic embryos, which may be able to benefit for capturing "naïve" pluripotency in vitro through inhibiting "primed" pluripotency.
Transcriptional regulation is a complex process that is controlled by a variety of factors, including enhancers and silencers. Silencers, also known as repressor elements, play a crucial role in the fine-tuning of gene expression by inhibiting or suppressing transcription in the human genome. Although significant progresses have been made, genome-wide silencer research is still in its early stages. Here, we used a genome-wide method called massively parallel reporter assays (MPRAs) to identify silencers in three human cell lines: K562, LNCap, and HEK293T. We identified 739,434, 643,484, and 491,952 silencer regions in these cell lines, respectively. We found that most of the silencers we identified had inhibitory activity and significantly enriched inhibitory motifs. These results confirm that silencers are ubiquitous in the human genome and play an important role in regulating gene expression. Therefore, our study provides a general strategy for genome-wide functional identification of silencer elements. This information could be used to better understand the mechanisms of gene regulation and to develop new therapeutic strategies for diseases that are caused by dysregulation of gene expression.
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