Medical Genetics, Medical University Vienna, Vienna, Austria.
Nat Protoc. 2010 Jun;5(6):1081-95. doi: 10.1038/nprot.2010.74. Epub 2010 May 20.
Human amniotic fluid stem cells (hAFSCs) are a very promising new type of fetal stem cells with numerous applications for basic science and cell-based therapies. They harbor a high differentiation potential and a low risk for tumor development, can be grown in large quantities and do not raise the ethical concerns associated with embryonic stem cells. RNA interference (RNAi) is a powerful technology to explain specific gene functions and has important implications for the clinical usage of tissue engineering. We provide a straightforward, 72-h-long protocol for siRNA-mediated gene silencing in hAFSCs. The lipid-based forward transfection protocol described in this article is the first RNAi approach for prolonged gene knockdown in hAFSCs. This protocol allows efficient, functional and reproducible gene knockdown in human stem cells over a prolonged period of time (approximately 2 weeks). We also show the successful use of this protocol in primary nontransformed nonimmortalized fibroblasts, cervical adenocarcinoma cells, transformed embryonic kidney cells, immortalized endometrial stromal cells and acute monocytic leukemia cells, suggesting a wide spectrum of applications in various cell types.
人羊膜干细胞（hAFSCs）是一种极具应用前景的新型胎儿干细胞，在基础科学和基于细胞的治疗中有广泛的应用。它们具有高分化潜能和低肿瘤发生风险，可以大量培养，且不存在与胚胎干细胞相关的伦理问题。RNA 干扰（RNAi）是一种强大的技术，可以用来解释特定基因的功能，对于组织工程的临床应用具有重要意义。我们提供了一种简单、持续 72 小时的 hAFSCs 中 siRNA 介导的基因沉默方案。本文描述的基于脂质的正向转染方案是 hAFSCs 中用于长时间基因敲低的第一个 RNAi 方法。该方案允许在较长时间内（约 2 周）对人干细胞进行高效、功能和可重复的基因敲低。我们还展示了该方案在原代非转化非永生化成纤维细胞、宫颈腺癌细胞、转化的胚胎肾细胞、永生化子宫内膜基质细胞和急性单核细胞白血病细胞中的成功应用，提示该方案在各种细胞类型中有广泛的应用。
