Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria
| Autor: | Peter A. Thomason, David A. Knecht, Douwe M. Veltman, Thomas D. Williams, Robert R. Kay, Robert H. Insall, Peggy Paschke, Jonathan R. Chubb |
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| Rok vydání: | 2019 |
| Předmět: |
0303 health sciences
Cell signaling General Immunology and Microbiology biology ved/biology General Chemical Engineering General Neuroscience Cellular differentiation fungi ved/biology.organism_classification_rank.species Mutagenesis (molecular biology technique) Transfection biology.organism_classification Dictyostelium General Biochemistry Genetics and Molecular Biology Dictyostelium discoideum Cell biology 03 medical and health sciences 0302 clinical medicine 030220 oncology & carcinogenesis Model organism Selectable marker 030304 developmental biology |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| Popis: | Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other important cellular biology questions. The technologies available to genetically manipulate Dictyostelium cells are well-developed. Transfections can be performed using different selectable markers and marker re-cycling, including homologous recombination and insertional mutagenesis. This is supported by a well-annotated genome. However, these approaches are optimized for axenic cell lines growing in liquid cultures and are difficult to apply to non-axenic wild-type cells, which feed only on bacteria. The mutations that are present in axenic strains disturb Ras signaling, causing excessive macropinocytosis required for feeding, and impair cell migration, which confounds the interpretation of signal transduction and chemotaxis experiments in those strains. Earlier attempts to genetically manipulate non-axenic cells have lacked efficiency and required complex experimental procedures. We have developed a simple transfection protocol that, for the first time, overcomes these limitations. Those series of large improvements to Dictyostelium molecular genetics allow wild-type cells to be manipulated as easily as standard laboratory strains. In addition to the advantages for studying uncorrupted signaling and motility processes, mutants that disrupt macropinocytosis-based growth can now be readily isolated. Furthermore, the entire transfection workflow is greatly accelerated, with recombinant cells that can be generated in days rather than weeks. Another advantage is that molecular genetics can further be performed with freshly isolated wild-type Dictyostelium samples from the environment. This can help to extend the scope of approaches used in these research areas. |
| Databáze: | OpenAIRE |
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