Paschke, P;
Knecht, DA;
Williams, TD;
Thomason, PA;
Insall, RH;
Chubb, JR;
Kay, RR;
(2019)
Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria.
Journal of Visualized Experiments
(143)
, Article e58981. 10.3791/58981.
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Abstract
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.
| Type: | Article |
|---|---|
| Title: | Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria |
| Location: | United States |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.3791/58981 |
| Publisher version: | http://dx.doi.org/10.3791/58981 |
| Language: | English |
| Additional information: | © 2019 Creative Commons Attribution 3.0 License (https://creativecommons.org/licenses/by/3.0/). |
| Keywords: | Genetics, Issue 143, Dictyostelium, transfection, knock-out, knock-in, extrachromosomal plasmids, act5, overexpression, chemotaxis, motility, macropinocytosis |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Lab for Molecular Cell Bio MRC-UCL |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10067640 |
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