Franz Alexandra, Brunner Erich, Basler Konrad
a Institute of Molecular Life Sciences, University of Zurich , Zurich , Switzerland.
Fly (Austin). 2017 Oct 2;11(4):303-311. doi: 10.1080/19336934.2017.1372068. Epub 2017 Aug 30.
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 Drosophila cell lines has remained a longstanding challenge, hampered by the difficulty of getting Drosophila cells to grow at low densities. Here, we describe a highly efficient workflow to generate clonal Cas9-engineered Drosophila 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 armadillo mutant Drosophila cell lines. Our method provides a powerful and simple workflow that improves the utility of Drosophila cells for genetic studies with CRISPR/Cas9.
多功能CRISPR/Cas9工具的最新发展显著提高了生成转基因动物和细胞系的便利性。然而，虽然对于哺乳动物细胞系来说，分离同基因细胞群体通常很简单，但克隆果蝇细胞系的生成一直是一项长期挑战，因为果蝇细胞在低密度下生长困难。在这里，我们描述了一种高效的工作流程，通过结合细胞池、在条件培养基中进行有限稀释以及使用等位基因特异性引物进行PCR，来生成克隆的Cas9工程果蝇细胞系，从而能够高效选择具有合适突变谱的克隆细胞系。我们通过记录八个独立的Cas9编辑的犰狳突变果蝇细胞系的分离、选择和验证来验证该方案。我们的方法提供了一个强大而简单的工作流程，提高了果蝇细胞在CRISPR/Cas9基因研究中的实用性。
