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

PloS One
Peggy PaschkeDouwe M Veltman

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 ...Continue Reading

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Citations

Jan 9, 2019·Journal of Cell Science·Thomas D WilliamsRobert R Kay
Mar 21, 2019·Development·Vlatka AntolovićJonathan R Chubb
Apr 13, 2019·Development·Agnes MiermontJonathan R Chubb
Apr 8, 2020·ELife·John Me NicholsJonathan R Chubb
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Apr 11, 2019·Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences·Jason S King, Robert R Kay
Mar 13, 2020·Proceedings of the National Academy of Sciences of the United States of America·Sven FlemmingCarsten Beta
Apr 2, 2020·Nucleic Acids Research·Peter KundertGad Shaulsky
Jan 16, 2019·Cells·Tetsuya MuramotoTakefumi Kawata
Apr 8, 2020·Biochemistry and Biophysics Reports·Christopher J PerryRobin S B Williams
Jun 30, 2021·Genome Research·Mariko Katoh-KurasawaGad Shaulsky
May 28, 2021·ELife·Ashley L ArthurMargaret A Titus
Aug 26, 2021·BMC Biology·Nicole GruenheitChristopher R L Thompson

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Methods Mentioned

BETA
transfection
PCR
motility assay
flow cytometry
fluorescence microscopy
transfections
gene knock-outs
transfect
confocal microscopy
FRET

Software Mentioned

Fiji
mScarlet
Zen2010
MOPS
MES

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