Paschke, P;
Knecht, DA;
Silale, A;
Traynor, D;
Williams, TD;
Thomason, PA;
Insall, RH;
... Veltman, DM; + view all
(2018)
Rapid and efficient genetic engineering of both wild type and axenic strains of Dictyostelium discoideum.
PLoS ONE
, 13
(5)
, Article e0196809.. 10.1371/journal.pone.0196809.
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Abstract
Dictyostelium has a mature technology for molecular-genetic manipulation based around transfection using several different selectable markers, marker re-cycling, homologous recombination and insertional mutagenesis, all supported by a well-annotated genome. However this technology is optimized for mutant, axenic cells that, unlike non-axenic wild type, can grow in liquid medium. There is a pressing need for methods to manipulate wild type cells and ones with defects in macropinocytosis, neither of which can grow in liquid media. Here we present a panel of molecular genetic techniques based on the selection of Dictyostelium transfectants by growth on bacteria rather than liquid media. As well as extending the range of strains that can be manipulated, these techniques are faster than conventional methods, often giving usable numbers of transfected cells within a few days. The methods and plasmids described here allow efficient transfection with extrachromosomal vectors, as well as chromosomal integration at a ‘safe haven’ for relatively uniform cell-to-cell expression, efficient gene knock-in and knock-out and an inducible expression system. We have thus created a complete new system for the genetic manipulation of Dictyostelium cells that no longer requires cell feeding on liquid media.
| Type: | Article |
|---|---|
| Title: | Rapid and efficient genetic engineering of both wild type and axenic strains of Dictyostelium discoideum |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1371/journal.pone.0196809 |
| Publisher version: | https://doi.org/10.1371/journal.pone.0196809 |
| Language: | English |
| Additional information: | This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ |
| Keywords: | Science & Technology, Multidisciplinary Sciences, Science & Technology - Other Topics, SOCIAL AMEBA, CELL-MOVEMENT, MACROPINOCYTOSIS, CHEMOTAXIS, EXPRESSION, DYNAMICS, PROTEIN, TRANSFORMATION, ESTABLISHMENT, EVOLUTION |
| 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/10052581 |
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