Generation of genome-modified Drosophila cell lines using SwAP. Issue 4 (2nd October 2017)

• Record Type: Journal Article
• Authors: Franz, Alexandra; Brunner, Erich; Basler, Konrad
• Is Part Of: Fly. Volume 11:Issue 4(2017)
• Page Start: 303
• Page End: 311
• Publication Date: 2017-10-02
• Subjects: Drosophila cells -- single cell cloning -- CRISPR/Cas9 -- allelespecific primer; Drosophila -- Periodicals; 595.77
• DOI: 10.1080/19336934.2017.1372068
• Languages: English
• ISSNs: 1933-6934

Abstract

ABSTRACT: 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.