Generation of genome-modified <i>Drosophila</i> cell lines using SwAP
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The ease of generating genetically modified animals and cell lines has been markedly increased by the recent development of the versatile CRISPR/Cas9 tool. However, while the isolation of isogenic cell populations is usually straightforward for mammalian cell lines, the generation of clonal <i>Drosophila</i> cell lines has remained a longstanding challenge, hampered by the difficulty of getting <i>Drosophila</i> cells to grow at low densities. Here, we describe a highly efficient workflow to generate clonal Cas9-engineered <i>Drosophila</i> cell lines using a combination of cell pools, limiting dilution in conditioned medium and PCR with allele-specific primers, enabling the efficient selection of a clonal cell line with a suitable mutation profile. We validate the protocol by documenting the isolation, selection and verification of eight independently Cas9-edited <i>armadillo</i> mutant <i>Drosophila</i> cell lines. Our method provides a powerful and simple workflow that improves the utility of <i>Drosophila</i> cells for genetic studies with CRISPR/Cas9.
近年来，多功能CRISPR/Cas9工具（CRISPR/Cas9）的研发显著提升了基因修饰动物与细胞系的构建效率与便捷性。然而，尽管哺乳动物细胞系的同基因细胞群分离通常较为简便，但克隆型果蝇（Drosophila）细胞系的构建长期以来都是一项棘手难题，其根源在于果蝇细胞在低密度条件下难以存活与增殖。本研究报道了一套高效实验流程，通过联合运用细胞池培养、条件培养基有限稀释法以及等位基因特异性引物PCR技术，实现经Cas9编辑的克隆型果蝇细胞系的构建，可高效筛选获得携带适配突变特征的克隆细胞系。本研究通过分离、筛选并验证8株独立构建的Cas9编辑型armadillo基因（armadillo）突变果蝇细胞系，对该实验方案的有效性进行了验证。本方法提供了一套简便高效的实验流程，可提升果蝇细胞结合CRISPR/Cas9工具开展遗传学研究的应用价值。
