1. National Engineering Research Center for Breeding Swine Industry, College of Animal Science of South China Agricultural University, Guangzhou 510642, China.
2. National & Local Joint Engineering Research Center for Breeding Animal Industry, Guangzhou 510642, China.
Yi Chuan. 2023 Jan 20;45(1):67-77. doi: 10.16288/j.yczz.22-244.
The abnormal modification of histone is an important factor restricting development of porcine cloned embryos. Overexpression of histone H3K9me3 demethylase KDM4 family can effectively improve the developmental efficiency of cloned embryos. In order to explore the effects of overexpression of H3K9me3 demethylase on the development of porcine cloned embryos, mRNA and mRNA were injected respectively into porcine cloned embryos at the 1-cell stage and 2-cell stage to detect the blastocyst rate; 2-cell stage cloned embryos injected with mRNA and embryo injection water (the control group) at the 1-cell stage were collected to detect the expression level of H3K9me3, and 4-cell stage cloned embryos were collected for single cell transcriptome sequencing, then the sequencing data was analyzed with KEGG and GO. The results showed that the blastocyst rate of porcine cloned embryos injected with mRNA at 1-cell stage was significantly higher than that of the control group (25.32 ± 0.74% 14.78 ± 0.87%), while cloned embryos injected with mRNA had a similar blastocyst rate with cloned embryos in control group (16.27 ± 0.77% 14.78 ± 0.87%). Porcine cloned embryos injected with mRNA and mRNA at 2-cell stage had a similar blastocyst rate with cloned embryos in control group (32.18 ± 1.67%, 30.04 ± 0.91% 31.22 ± 1.40%). The expression level of H3K9me3 in cloned embryos injected with mRNA at 1-cell stage was lower than that in control group. There were 133 differentially expressed genes detected by transcriptome sequencing, including 52 up-regulated genes and 81 down-regulated genes. Pathways enriched by GO analyses were mainly related to protein localization. Pathways enriched by KEGG analyses were related to cellular senescence and acute myeloid leukemia. These results suggest that overexpression of histone H3K9me3 demethylase KDM4A can significantly improve the developmental efficiency of porcine cloned embryos.
组蛋白的异常修饰是限制猪克隆胚胎发育的重要因素。过表达组蛋白 H3K9me3 去甲基化酶 KDM4 家族可以有效提高克隆胚胎的发育效率。为了探讨 H3K9me3 去甲基化酶过表达对猪克隆胚胎发育的影响，分别将 mRNA 和 mRNA 注射到猪克隆胚胎的 1 细胞期和 2 细胞期，检测囊胚率；收集 1 细胞期注射 mRNA 和胚胎注射水（对照组）的 2 细胞期克隆胚胎，检测 H3K9me3 的表达水平，收集 4 细胞期克隆胚胎进行单细胞转录组测序，然后对测序数据进行 KEGG 和 GO 分析。结果表明，1 细胞期注射 mRNA 的猪克隆胚胎囊胚率显著高于对照组（25.32±0.74% 14.78±0.87%），而注射 mRNA 的克隆胚胎囊胚率与对照组相似（16.27±0.77% 14.78±0.87%）。2 细胞期注射 mRNA 和 mRNA 的猪克隆胚胎囊胚率与对照组相似（32.18±1.67%、30.04±0.91% 31.22±1.40%）。1 细胞期注射 mRNA 的克隆胚胎 H3K9me3 的表达水平低于对照组。转录组测序共检测到 133 个差异表达基因，其中上调基因 52 个，下调基因 81 个。GO 分析富集的通路主要与蛋白质定位有关。KEGG 分析富集的通路与细胞衰老和急性髓系白血病有关。这些结果表明，组蛋白 H3K9me3 去甲基化酶 KDM4A 的过表达可以显著提高猪克隆胚胎的发育效率。
