Developmental abnormalities associated with the cloning process suggest that reprogramming of donor nuclei into an embryonic state may not be fully completed in most of the cloned animals. One of the areas of interest in this regard, is the analysis of gene expression patterns in nuclear transfer (NT) embryos to dissect the processes that failed and develop means to overcome the limitations imposed by these factors. In this study, we investigated expression patterns of histone deacetylase-1, -2, -3 (HDAC-1, -2, -3), DNA methyltransferase-3a (DNMT3A), and octamer binding protein-4 gene (OCT4) in donor cells with different cloning efficiencies and NT embryos derived from these cells employing a real-time RT-PCR assay. All genes investigated followed altered expression patterns in NT embryos when compared to IVF-derived embryos. In general, expression of HDAC genes was elevated especially at the compact morula stage and comparable to in vitro fertilized (IVF) embryos at the hatched blastocyst stage. DNMT3A expression in NT embryos was lower than IVF embryos at all stages. Oct-4 transcript levels were also reduced in cloned compared to IVF embryos at the compact morula and blastocyst stages. This difference disappeared at the hatched blastocyst stage. There was a donor cell effect on the expression patterns of all genes investigated. These results demonstrate altered gene expression patterns for certain genes, in cloned cattle embryos from our donor cells of different efficiency in producing live offspring. Therefore we suggest that differences in expression of developmentally important genes during early embryo development may characterize the efficiency of donor cells in producing live offspring.

译文

与克隆过程相关的发育异常表明,在大多数克隆动物中,供体核重编程为胚胎状态可能尚未完全完成。在这方面,感兴趣的领域之一是分析核转移 (NT) 胚胎中的基因表达模式,以剖析失败的过程,并开发克服这些因素所施加限制的手段。在这项研究中,我们研究了组蛋白deacetylase-1,-2,-3 (HDAC-1,-2,-3),DNA methyltransferase-3a (DNMT3A),和八聚体结合蛋白4基因 (OCT4) 在具有不同克隆效率的供体细胞中,以及使用实时rt-pcr分析从这些细胞衍生的NT胚胎。与IVF衍生的胚胎相比,所研究的所有基因均遵循NT胚胎中表达模式的改变。通常,HDAC基因的表达升高,尤其是在紧凑的桑ula阶段,与孵化的胚泡阶段的体外受精 (IVF) 胚胎相当。在所有阶段,NT胚胎中DNMT3A的表达均低于IVF胚胎。与紧凑型桑ula和胚泡期的IVF胚胎相比,克隆的10月-4转录本水平也降低。这种差异在孵化的胚泡阶段消失了。对所有研究基因的表达模式都有供体细胞影响。这些结果表明,在来自我们的供体细胞的克隆牛胚胎中,某些基因的基因表达模式发生了改变,这些基因在生产后代方面效率不同。因此,我们建议在早期胚胎发育过程中发育重要基因表达的差异可能是供体细胞产生活后代的效率的特征。

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