通过快速选择细胞池对内源基因进行功能编辑（在果蝇卵巢体腔体细胞中快速生成内源标记基因）。
Functional editing of endogenous genes through rapid selection of cell pools (Rapid generation of endogenously tagged genes in Drosophila ovarian somatic sheath cells).
机构信息
National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA.
Department of Biochemistry, School of Medicine, University of Patras, 26504 Patras, Greece.
出版信息
Nucleic Acids Res. 2022 Aug 26;50(15):e90. doi: 10.1093/nar/gkac448.
The combination of genome-editing and epitope tagging provides a powerful strategy to study proteins with high affinity and specificity while preserving their physiological expression patterns. However, stably modifying endogenous genes in cells that do not allow for clonal selection has been challenging. Here, we present a simple and fast strategy to generate stable, endogenously tagged alleles in a non-transformed cell culture model. At the example of piwi in Drosophila ovarian somatic sheath cells, we show that this strategy enables the generation of an N-terminally tagged protein that emulates the expression level and subcellular localization of the wild type protein and forms functional Piwi-piRNA complexes. We further present a concise workflow to establish endogenously N-terminally and C-terminally tagged proteins, and knockout alleles through rapid selection of cell pools in fly and human models.
基因组编辑和表位标记的结合为研究具有高亲和力和特异性的蛋白质提供了一种强大的策略，同时保持其生理表达模式。然而，在不允许克隆选择的细胞中稳定修饰内源性基因一直具有挑战性。在这里，我们提出了一种简单快速的策略，可在非转化细胞培养模型中生成稳定的内源性标记等位基因。以果蝇卵巢体细胞鞘细胞中的 piwi 为例，我们表明该策略能够产生 N 端标记的蛋白质，该蛋白质模拟野生型蛋白质的表达水平和亚细胞定位，并形成功能性 Piwi-piRNA 复合物。我们进一步提出了一个简洁的工作流程，通过在果蝇和人类模型中快速选择细胞池，建立内源性 N 端和 C 端标记蛋白和敲除等位基因。
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