Department of Medical Microbiology, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, Nijmegen, The Netherlands.
Methods Mol Biol. 2022;2509:3-22. doi: 10.1007/978-1-0716-2380-0_1.
In insects, PIWI-interacting (pi)RNAs fulfill versatile regulatory functions inside and outside the germline, including posttranscriptional repression of transposable elements and regulation of gene expression. Canonically, piRNAs act-and have been studied-as a conglomerate of several thousand sequences that cooperatively silence target RNAs. Interestingly, however, an increasing number of studies have demonstrated that individual piRNAs can have profound biological activity as a unique piRNA sequence. Prime examples are the tapiR1 and 2 piRNAs, which mediate target RNA degradation in the developing embryo of Aedes mosquitoes. To study such outstanding individual piRNA species, we describe here a method to interfere with RNA target silencing using antisense oligonucleotides in cell culture as well as in mosquito pre-blastoderm embryos. Although the method has been established for Aedes mosquitoes, it can likely be adapted for use in other invertebrate species as well.
在昆虫中，PIWI 相互作用（pi）RNAs 在生殖细胞内外发挥多种调节功能，包括转座元件的转录后抑制和基因表达的调控。通常，piRNAs 作为几千个序列的集合起作用，并协同沉默靶 RNA。然而，有趣的是，越来越多的研究表明，单个 piRNAs 可以作为独特的 piRNA 序列发挥深远的生物学活性。一个突出的例子是来自埃及伊蚊胚胎的 tapiR1 和 2 piRNAs，它们介导靶 RNA 的降解。为了研究这种杰出的单个 piRNA 种类，我们在这里描述了一种在细胞培养以及蚊子原肠胚胚胎中使用反义寡核苷酸干扰 RNA 靶标沉默的方法。尽管该方法已在埃及伊蚊中建立，但很可能适用于其他无脊椎动物物种。
