Generation of genome-modified Drosophila cell lines using SwAP
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 Drosophi...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2017-10-01
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| Series: | Fly |
| Subjects: | |
| Online Access: | http://dx.doi.org/10.1080/19336934.2017.1372068 |
| _version_ | 1797678377860071424 |
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| author | Alexandra Franz Erich Brunner Konrad Basler |
| author_facet | Alexandra Franz Erich Brunner Konrad Basler |
| author_sort | Alexandra Franz |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-11T22:58:50Z |
| format | Article |
| id | doaj.art-a2fc17f691254be0a2708bf0ea06f63e |
| institution | Directory Open Access Journal |
| issn | 1933-6934 1933-6942 |
| language | English |
| last_indexed | 2024-03-11T22:58:50Z |
| publishDate | 2017-10-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | Fly |
| spelling | doaj.art-a2fc17f691254be0a2708bf0ea06f63e2023-09-21T15:09:08ZengTaylor & Francis GroupFly1933-69341933-69422017-10-0111430331110.1080/19336934.2017.13720681372068Generation of genome-modified Drosophila cell lines using SwAPAlexandra Franz0Erich Brunner1Konrad Basler2Institute of Molecular Life Sciences, University of ZurichInstitute of Molecular Life Sciences, University of ZurichInstitute of Molecular Life Sciences, University of ZurichThe 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.http://dx.doi.org/10.1080/19336934.2017.1372068drosophila cellssingle cell cloningcrispr/cas9allelespecific primer |
| spellingShingle | Alexandra Franz Erich Brunner Konrad Basler Generation of genome-modified Drosophila cell lines using SwAP Fly drosophila cells single cell cloning crispr/cas9 allelespecific primer |
| title | Generation of genome-modified Drosophila cell lines using SwAP |
| title_full | Generation of genome-modified Drosophila cell lines using SwAP |
| title_fullStr | Generation of genome-modified Drosophila cell lines using SwAP |
| title_full_unstemmed | Generation of genome-modified Drosophila cell lines using SwAP |
| title_short | Generation of genome-modified Drosophila cell lines using SwAP |
| title_sort | generation of genome modified drosophila cell lines using swap |
| topic | drosophila cells single cell cloning crispr/cas9 allelespecific primer |
| url | http://dx.doi.org/10.1080/19336934.2017.1372068 |
| work_keys_str_mv | AT alexandrafranz generationofgenomemodifieddrosophilacelllinesusingswap AT erichbrunner generationofgenomemodifieddrosophilacelllinesusingswap AT konradbasler generationofgenomemodifieddrosophilacelllinesusingswap |