Rapid and efficient genetic engineering of both wild type and axenic strains of <i>Dictyostelium discoideum</i>

<div><p><i>Dictyostelium</i> 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 <i>Dictyostelium</i> 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 <i>Dictyostelium</i> cells that no longer requires cell feeding on liquid media.</p></div

Selection and transfection conditions for bacterially-grown cells.

<p>(A) Antibiotic sensitivity of bacterially-grown cells. Cells growing in petri dishes with <i>K</i>. <i>pneumoniae</i> bacteria in SorMC buffer were treated with the indicated antibiotics. Individual wells were harvested after 36 and 84 hours and the cells plated clonally on SM agar plates to determine viability (error bars are SD). (B) Schematic overview of the extrachromosomal expression vector, with the four modules shown in different colouring: green = resistance marker; blue = expression cassette; purple = <i>Dictyostelium</i> replication module; yellow = <i>E</i>. <i>coli</i> replication module. (C-D) The efficiencies of different promoters at driving the selectable marker in bacterially-cultured cells. Cells were transfected with 1 μg of respective plasmid and the number of colonies counted after 2 days (n = 3; error bars are SD). (E-F) Optimization of electroporation conditions. The survival of cells and number of transfectants obtained after electroporation with 2 square waves, 1 second apart, at the indicated voltages and pulse lengths. Survival was determined by clonal plating on SM agar. (H) Table of extrachromosmal plasmids in the new pDM/pPI system, including inducible and shuttle vectors. (I) Workflow of a non-axenic transfection using an extrachromosomal expression plasmid. If not indicated otherwise, NC4 cells were used in all experiments.</p

Knock-ins to the endogenous locus generated with cells grown in bacterial suspension.

<p>Knock-ins to the endogenous locus generated with cells grown in bacterial suspension.</p

Generation of gene knock-outs.

<p>(A) Scheme of the knock out resistance cassette. The resistance marker is flanked by loxP sites (yellow triangles). A 20 bp custom primer site on each end of the cassette allows assembly of the knock-out construct by joined PCR (for a detailed protocol see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196809#pone.0196809.s018" target="_blank">S2 File</a>). The resistance marker is driven by the <i>coaA</i> promotor (green) and terminated using the <i>mhcA</i> terminator (pink). (B) Map of a knock-out plasmid (HygR). Promotors are displayed in green, terminators in pink, open reading frames in blue and origins of replication are highlighted in grey. (C) Knock-out transfection efficiency showing the percentage of correct clones plotted against the number of clones obtained after transfection. The different wild type strains used are indicated through different colouring (AX2 in blue, DdB in yellow and NC4 in green (D) Map of a Cre expression plasmid used to remove the resistance cassette flanked by LoxP sites from mutants. Promoters are displayed in green, terminators in pink, open reading frames in blue and origins of replication are highlighted in grey. D1, D5 and G5 are the minimal set of ORFs necessary to keep the plasmid extrachromosomal.</p

The relationship between axenic growth, cell motility and macropinocytosis and the role of RasS in vegetative cells.

<p>(A) Cell tracks of randomly moving vegetative cells. AX2 cells were grown in HL5 or bacterial suspension. DdB and NC4 cells were cultured in bacterial suspension. Pictures show the tracks of 20 individual cells for each strain. The cells were filmed for 30 minutes at 2 frames per minute. (B) The effect of axenic mutations and axenic growth on cell motility. AX2 cells grown in HL5 move more slowly than AX2 grown on bacteria, which in turn are slower than wild type DdB or NC4 cells. Movies were taken at 2 frames/minute, with 3 movies per strain and 20 cells analysed from each movie. Whiskers show maximal and minimal values, with the median shown as a solid black line inside each box, where the top and the bottom represent the first and third quartiles. (C) The actin cytoskeleton of vegetative AX2, DdB and NC4 cells. Macropinosomes are the dominant feature of AX2 cells grown in HL5, whereas pseudopods dominate DdB and NC4 cells grown on bacteria; AX2 cells grown on bacteria are intermediate between these extremes. Cells expressing LifeAct-GFP as a reporter for F-actin were cultured in HL5 or bacterial suspension and examined by confocal microscopy. Scale bar 10 μm. (D) Cloning strategy used for the knock-out of <i>ras</i>S. The gene is shown in yellow and the disruption cassette coding for the hygromycin resistance gene in orange. The recombination arms highlighted in grey are 700 bp each and cover just about 30 bp of the coding sequence of <i>ras</i>S gene on each side. Primer sites are indicated with arrows. (E) Confirmation by PCR of two independent <i>rasS-</i> knock-out mutants (HM1920 <i>ras</i>S- 1–1 and HM1921 <i>ras</i>S- 3–1) using two different primer combinations. The band shift in first PCR (P1/P2) shows that the gene was successfully disrupted, with no wild type band detectable. The second PCR (P3/P2) confirms the correct insertion of the resistance cassette in the <i>ras</i>S gene. DdB wild type DNA was used as control in both reactions. The binding site of Primer 2 is located outside of the 3’ recombination arm and so is locus specific. (F-G) Verification of the disruption of the <i>ras</i>S genes by Southern Blotting. Cleanly disrupted recombinants are revealed by the loss of the wild type 1.6 kb band on Southern blots hybridised to a ³²P labelled <i>ras</i>S probe. The appearance of a band of approximately 3.2 kb indicates the genomic <i>ras</i>S locus has been successfully disrupted by the hygromycin resistance cassette (The blot has been cropped. The unmodified original is shown in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196809#pone.0196809.s018" target="_blank">S2 File</a>). (H) The speed of random movement of vegetative wild type (yellow) and two <i>rasS-</i> mutants (orange) was measured as described in Fig 5B. The average of three experiments; maximal and the minimal values are displayed by whiskers and the median by the black line inside each box plot. Statistical analysis used an unpaired two-tailed student-t test (** p <0.005, *** p<0.0005). (G) Fluid uptake by DdB and <i>rasS-</i> mutant cells. The cells were incubated over night in HL5 containing 10% foetal calf serum, then the uptake of TRITC-dextran measured after 60 minutes by flow cytometry. Results are normalised to DdB. The error bars indicate the SD, n = 6; statistical analysis by an unpaired two-tailed Student-t test (* p <0.005, ** p<0.0005).</p

Gene knock-outs generated with cells grown in bacterial suspension.

<p>Gene knock-outs generated with cells grown in bacterial suspension.</p

Culturing of cells on bacteria.

<p>(A) DIC images of AX2 cells cultured axenically in HL5 medium and of NC4 cells cultured in a suspension of <i>K</i>. <i>aerogenes</i> bacteria. A disadvantage of growth with bacteria is that bacteria obscure the <i>Dictyostelium</i> cells. (B) <i>Dictyostelium</i> cells grow much faster on bacteria than in HL5 medium. Cells were grown in tissue-culture dishes with the food source indicated. (C) The adhesion of <i>Dictyostelium</i> cells is impaired by co-culturing with <i>E</i>. <i>coli</i>. Cells in 9 cm Petri dishes were shaken in 10 ml of SorMC buffer supplemented with bacteria to an OD₆₀₀ of 2 on a rotary shaker at the speeds indicated for 30 minutes. The medium was then collected, the cells spun down, resuspended and counted. The cells remaining on the plate were rinsed off with SorMC, spun down, resuspended and counted. Error bars show mean and SD of at least 3 experiments.</p

Knock-ins to the <i>act5</i> locus generated with cells grown in bacterial suspension.

<p>Knock-ins to the <i>act5</i> locus generated with cells grown in bacterial suspension.</p

Stable expression using integrating plasmids.

<p>(A) Schematic map of the REMI expression vector and table of versions for N-terminal and C-terminal tagging. In all plasmid maps the promoters are highlighted in green, terminators in pink, open reading frames in blue and origins of replication in grey. (B) Map of the <i>act5</i> integration plasmid including a table of all different versions available for the creation of fluorescent fusion proteins. The recombination arms used for the integration at the <i>act5</i> locus are highlighted in orange. (C) Map and schematic overview of the plasmid for targeted in-frame integration to produce C-terminal knock-ins. The crossover sequences used for the knock-in construct assembly are highlighted in green. (D-F) Efficiency of transfection using the three different strategies for making stable cell lines. Cells were transfected with 1–2 μg DNA using the optimised method outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196809#pone.0196809.g002" target="_blank">Fig 2</a>. For the <i>act5</i> and targeted in-frame integrations the percentage of positive clones is given on the x-axis. The different strains used for the transfection are separately shown indicated by different colouring (AX2 in blue, DdB in yellow and NC4 in green). See Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196809#pone.0196809.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196809#pone.0196809.t002" target="_blank">2</a> for details of the genes. White circles shown for the REMI efficiency are for NC4. Best results came with 2 μg of <i>Bam</i>HI-digested DNA, 5 units of <i>Dpn</i>II and a vector with the <i>coa</i>A promoter replaced by the <i>act14</i> promoter. (G) Table summing up the advantages and disadvantages of the three different integration methods.</p

Chemotaxis of cells from different melanoma stages.

<p>(A) Chemotaxis of a panel of six cell lines from different melanoma stages (RGP, green; VGP, purple; metastatic, red) up a 0%–10% FBS gradient was measured as above (<i>n</i>≥45 cells per cell line). (B) Chemotactic index of cells from different stages. Data from (A) were collated by melanoma stage. Chemotaxis improves as the stage of melanoma progresses, although even the earliest RGP cells show clear chemotaxis. (C) Speeds of cells from different stages. Data from (A) were collated by melanoma stage. Metastatic lines are conspicuously faster (<i>p</i>-values from unpaired <i>t</i>-tests), although again the speed of RGP and VGP cells is still relatively high for non-haematopoietic cells.</p