Geisel School of Medicine at Dartmouth, Department of Molecular and Systems Biology, Hanover, NH, USA.
University of North Carolina, Department of Biology, Chapel Hill, NC, USA.
Fungal Genet Biol. 2021 Nov;156:103615. doi: 10.1016/j.fgb.2021.103615. Epub 2021 Aug 20.
Single molecule RNA-FISH (smFISH) is a valuable tool for analysis of mRNA spatial patterning in fixed cells that is underutilized in filamentous fungi. A primary complication for fixed-cell imaging in filamentous fungi is the need for enzymatic cell wall permeabilization, which is compounded by considerable variability in cell wall composition between species. smFISH adds another layer of complexity due to a requirement for RNase free conditions. Here, we describe the cloning, expression, and purification of a chitinase suitable for supplementation of a commercially available RNase-free enzyme preparation for efficient permeabilization of the Neurospora cell wall. We further provide a method for smFISH in Neurospora which includes a tool for generating numerical data from images that can be used in downstream customized analysis protocols.
单细胞 RNA-FISH（smFISH）是一种分析固定细胞中 mRNA 空间模式的有价值的工具，但在丝状真菌中未得到充分利用。丝状真菌固定细胞成像的一个主要难题是需要酶解细胞壁通透化，而不同物种之间细胞壁成分的差异很大，使得这一过程更加复杂。由于需要无 RNA 酶的条件，smFISH 又增加了一个复杂性。在这里，我们描述了一种几丁质酶的克隆、表达和纯化，该酶适用于补充市售的无 RNA 酶酶制剂，以有效通透 Neurospora 的细胞壁。我们进一步提供了一种在 Neurospora 中进行 smFISH 的方法，包括一种从图像生成数字数据的工具，可用于下游定制分析方案。
