Adams A M, Pratt S L, Stice S L
Animal and Dairy Science Department, University of Georgia, 425 River Road, Athens, Georgia 30602, USA.
Mol Reprod Dev. 2005 Nov;72(3):311-9. doi: 10.1002/mrd.20357.
RNA interference (RNAi) has rapidly developed into one of the most widely applied technologies in molecular and cellular research, and although young, is now an essential experimental tool. The versatility of RNAi, especially in mammalian species, lends to its potential applications in a wide array of fields. Without having to genetically manipulate the genome, the ability to selectively reduce the level of a specific transcript using small interfering RNA (siRNA) molecules has great appeal in studying reprogramming issues in somatic cell nuclear transfer (SCNT) embryos. In such embryos, the aberrant expression of the somatic isoform of Dnmt1 (Dnmt1s), the enzyme responsible for maintaining DNA methylation in all somatic cells, has been implicated as one factor in the improper reprogramming of the donor genome. In the present study, the ability to develop a method allowing for the knockdown, or reduction, of Dnmt1s in primary fibroblast cells, like those commonly used as karyoplast donors in SCNT studies, was investigated in primary murine and bovine fibroblast cells as well as in a compromised cell line (NIH/3T3). Two Dnmt1s-specific siRNA candidates were designed and tested. Using optimized conditions, these siRNAs were transiently transfected into the cells with total RNA and nuclear protein being collected. A 56.5% knockdown in Dnmt1s was achieved in the compromised and primary murine cells whereas Dnmt1s was reduced by 15.4% in the primary bovine cells. A reduction in Dnmt1s mRNA did not correspond to a reduction in protein as determined by immunodetection of Western blots. Overall, this study demonstrated the ability of siRNA to knockdown Dnmt1s mRNA in primary fibroblast donor cells. In order to substantially increase the efficiency while decreasing the anomalies seen in SCNT, novel techniques, like the one proposed, are needed to assist the oocyte's ability to reprogram a differentiated genome.
RNA干扰（RNAi）已迅速发展成为分子和细胞研究中应用最广泛的技术之一，尽管它尚年轻，但如今已成为一种必不可少的实验工具。RNAi的多功能性，尤其是在哺乳动物物种中的多功能性，使其在众多领域具有潜在应用价值。无需对基因组进行基因操作，利用小干扰RNA（siRNA）分子选择性降低特定转录本水平的能力，在研究体细胞核移植（SCNT）胚胎的重编程问题方面具有很大吸引力。在这类胚胎中，负责在所有体细胞中维持DNA甲基化的酶Dnmt1的体细胞异构体（Dnmt1s）的异常表达，被认为是供体基因组重编程不当的一个因素。在本研究中，研究了在原代小鼠和牛成纤维细胞以及受损细胞系（NIH/3T3）中，开发一种能够在原代成纤维细胞中敲低或降低Dnmt1s的方法的能力，这些原代成纤维细胞常用于SCNT研究中的核质体供体。设计并测试了两种Dnmt1s特异性siRNA候选物。在优化条件下，将这些siRNA瞬时转染到细胞中，并收集总RNA和核蛋白。在受损和原代小鼠细胞中，Dnmt1s实现了56.5%的敲低，而在原代牛细胞中，Dnmt1s降低了15.4%。通过蛋白质印迹免疫检测确定，Dnmt1s mRNA的减少与蛋白质的减少并不对应。总体而言，本研究证明了siRNA在原代成纤维细胞供体细胞中敲低Dnmt1s mRNA的能力。为了在降低SCNT中出现的异常情况的同时大幅提高效率，需要像所提出的这种新技术来协助卵母细胞对分化基因组进行重编程的能力。
