Center for Pregnancy and Newborn Research, Department of Obstetrics and Gynecology, University of Texas Health Science Center San Antonio, Mail Code 7836, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA.
Placenta. 2012 Oct;33(10):754-62. doi: 10.1016/j.placenta.2012.07.003. Epub 2012 Jul 23.
Studies in primary human trophoblasts provide critical insights into placental function in normal and complicated pregnancies. Mechanistic studies in these cells require experimental tools to modulate gene expression. Lipid-based methods to transfect primary trophoblasts are fairly simple to use and allow for the efficient delivery of nucleic acids, but potential toxic effects limit these methods. Viral vectors are versatile transfection tools of native trophoblastic or foreign cDNAs, providing high transfection efficiency, low toxicity and stable DNA integration into the trophoblast genome. RNA interference (RNAi), using small interfering RNA (siRNA) or microRNA, constitutes a powerful approach to silence trophoblast genes. However, off-target effects, such as regulation of unintended complementary transcripts, inflammatory responses and saturation of the endogenous RNAi machinery, are significant concerns. Strategies to minimize off-target effects include using multiple individual siRNAs, elimination of pro-inflammatory sequences in the siRNA construct and chemical modification of a nucleotide in the guide strand or of the ribose moiety. Tools for efficient gene targeting in primary human trophoblasts are currently available, albeit not yet extensively validated. These methods are critical for exploring the function of human trophoblast genes and may provide a foundation for the future application of gene therapy that targets placental trophoblasts.
在原发性人滋养层细胞中的研究为正常和复杂妊娠中的胎盘功能提供了重要的见解。这些细胞中的机制研究需要实验工具来调节基因表达。基于脂质的转染原发性滋养层细胞的方法非常简单易用，并允许高效地递送核酸，但潜在的毒性作用限制了这些方法的应用。病毒载体是天然滋养层或外源 cDNA 的多功能转染工具，提供了高转染效率、低毒性和稳定的 DNA 整合到滋养层基因组中。RNA 干扰 (RNAi)，使用小干扰 RNA (siRNA) 或 microRNA，是一种沉默滋养层基因的强大方法。然而，脱靶效应，如对意外互补转录物的调控、炎症反应和内源性 RNAi 机制的饱和，是一个重大的关注点。最小化脱靶效应的策略包括使用多个单独的 siRNA、消除 siRNA 构建体中的促炎序列以及在向导链或核糖部分的核苷酸上进行化学修饰。目前已经有高效的基因靶向在原发性人滋养层细胞中的工具，但尚未得到广泛验证。这些方法对于探索人类滋养层基因的功能至关重要，并可能为未来针对胎盘滋养层的基因治疗应用提供基础。
