Rapid and efficient genetic engineering of both wild type and axenic strains of Dictyostelium discoideum

Paschke, P., Knecht, D. A., Silale, A., Traynor, D., Williams, T. D., Thomason, P. A., Insall, R. H. , Chubb, J. R., Kay, R. R. and Veltman, D. M. (2018) Rapid and efficient genetic engineering of both wild type and axenic strains of Dictyostelium discoideum. PLoS ONE, 13(5), e0196809. (doi: 10.1371/journal.pone.0196809) (PMID:29847546) (PMCID:PMC5976153)

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.

• Item Type: Articles
• Additional Information: This work was funded by Medical Research Council grant MC_U105115237 to R. R. Kay, BBSRC (Biotechnology and Biological Sciences Research Council) grant BB/K009699/1 to R. R. Kay, Cancer Research UK grant A15672 to R. H. Insall, Wellcome Trust Senior Fellowship 202867/Z/16/Z to Jonathan R Chubb, and MRC funding (MC_U12266B) to the MRC LMCB University Unit at UCL.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Thomason, Dr Peter and Insall, Professor Robert and Knecht, Prof David

Creator Roles

Knecht, D. A.: Conceptualization, Investigation, Writing – review and editing
Thomason, P. A.: Investigation, Writing – review and editing
Insall, R. H.: Supervision, Writing – review and editing

• Authors: Paschke, P., Knecht, D. A., Silale, A., Traynor, D., Williams, T. D., Thomason, P. A., Insall, R. H., Chubb, J. R., Kay, R. R., and Veltman, D. M.
• College/School: College of Medical Veterinary and Life Sciences > School of Cancer Sciences
• Journal Name: PLoS ONE
• Publisher: Public Library of Science
• ISSN: 1932-6203
• ISSN (Online): 1932-6203
• Copyright Holders: Copyright © 2018 Paschke et al.
• First Published: First published in PLoS ONE 13(5): e0196809
• Publisher Policy: Reproduced under a Creative Commons License