Reprogramming of fibroblast nuclei after transfer into bovine oocytes.
Reprogramming of fibroblast nuclei after transfer into bovine oocytes.
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DOI：
10.1089/15204559950020102
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发表时间：
1999-01-01
期刊：
影响因子：
--
通讯作者：
Westhusin, M E
中科院分区：
文献类型：
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作者：
De Sousa, P A;Winger, Q;Westhusin, M E
最近在动物克隆方面取得的里程碑式的成就表明，细胞分化的过程原则上是可以逆转的。这种逆转必然需要大规模的基因重编程，对此我们所知甚少。在本研究中，我们描述了在以胎儿成纤维细胞作为核供体（FF NT）的核移植（NT）产生的牛胚胎中，囊胚期特异性mRNA表达的保守程度。将FF NT胚胎的mRNA库与胚胎卵裂球（Emb NT）构建的NT胚胎的mRNA库进行比较，并与体内或体外条件下的胚胎进行比较，最后与成纤维细胞进行比较。采用差异显示方法对比胚胎/细胞特异性mRNA库。随机寡核苷酸引物对组合被用于对mRNA群体进行细分，并将单个mRNA表示为100至800个碱基对的拷贝DNA （cDNA）带。无论牛囊胚是在体内还是体外发育，还是在胚胎卵裂球或胎儿成纤维细胞核移植后获得，它们的mRNA谱都高度保守，与胎儿成纤维细胞的mRNA谱不同。与体外培养囊胚相比，FF NT、Emb NT和体内培养囊胚的cDNA条带模式约有95%的保守性。相比之下，成纤维细胞的cDNA条带与体外来源的囊胚相比只有67%的保守性（p < 0.0001），这表明核移植诱导了基因转录的剧烈变化。核移植后，胎儿成纤维细胞中的基因表达被重新编程，以模仿胚胎着床前的发育。这些变化的未来特征将是非常宝贵的，以确定合适的细胞类型，作为胚胎重建的核供体，并提供信息，可用于提高克隆动物的核移植效率。
Recent landmark achievements in animal cloning have demonstrated that the events of cell differentiation can, in principle, be reversed. This reversal necessarily requires large-scale genetic reprogramming, of which little is known. In the present study we characterized the extent to which blastocyst stage-specific mRNA expression would be conserved in bovine embryos produced by nuclear transfer (NT) using fetal fibroblasts as nuclei donors (FF NT). The mRNA pool of FF NT embryos was compared with that of NT embryos reconstructed from embryonic blastomeres (Emb NT), with embryos produced under in vivo or in vitro conditions, and finally with fibroblast cells. Embryo/cell-specific mRNA pools were contrasted using differential display methodology. Random oligonucleotide primer pair combinations were used to subfractionate mRNA populations and represent individual mRNAs as copy DNA (cDNA) bands ranging in size from 100 to 800 base pairs. Regardless of whether bovine blastocysts developed in vivo or in vitro, or were derived after nuclear transplantation with embryonic blastomeres or fetal fibroblasts, their mRNA profile was highly conserved and distinct from that of fetal fibroblast cells. There was approximately 95% conservation in cDNA banding patterns between FF NT, Emb NT, and in vivo derived blastocysts, when compared with in vitro derived blastocysts. In contrast, the cDNA banding in fibroblasts was only 67% conserved with in vitro derived blastocysts (p < 0.0001), indicating that dramatic changes in gene transcription are induced by nuclear transplantation. After nuclear transplantation, gene expression in fetal fibroblasts is reprogrammed so to mimic that of preimplantation embryo development. Future characterization of these changes will be invaluable for the identification of suitable cell types to serve as nuclear donors for embryo reconstruction and provide information that can be used to improve the efficiency of cloning animals by nuclear transplantation.