The authors thank the Kay lab for input to this method and the LMB microscopy and flow cytometry facilities for excellent scientific and technical support. 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.

<ol>
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The authors have no conflicts of interest to disclose.

The use of non-axenic, wild-type Dictyostelium cells has been very limited so far in molecular research. Available methods for genetic engineering of these strains have lacked reliability and efficiency23, preventing their general adoption. The generated materials and protocols presented here can be used for any D. discoideum strain independent of its ability to grow in liquid medium. It should be mentioned that this protocol is optimized for NC4-derived cell lines. Transfection efficiencies for freshly isolated strains from the wild differ from NC4, as we have observed before and are shown here for V12M2 and WS21628,9. The electroporation conditions seem in particular to have a considerable influence on transfection efficiencies and may require further optimization for some strains. In general, a sufficient amount of transfectants have been observed in all strains tested so far, showing that these methods are workable. The number of positive transfectants obtained using NC4-derived strains is higher compared to other non-axenic wild-type strains, but in all cases, sufficient numbers of transfectants are obtained to allow for further experiments. This, plus the simplicity of our protocol, is the major improvement compared to earlier attempts8,9.
These new non-axenic protocols cover all standard genetic procedures and add further advantages, since transfections can be performed rapidly and efficiently in parallel. Positive clones can be obtained in days rather than weeks, since growth on bacteria halves the division time. The newly established plasmids also work under axenic conditions and can be routinely used under both growth conditions, which is another advancement of this methology22. As introduced in the protocol, the plasmid used for selection on bacteria have special requirements that are crucial for the success of the transfection. In particular, the promoters driving the expression and resistance cassettes are important for successful transfection. The often used act6 (actin 6) promoter24 for driving the resistance or expression cassettes lacks efficiency when cells are grown on bacteria. In our plasmid system, the highly active act15 (actin 15) promoter drives all expression cassettes, while the resistance cassettes are under the control of a coA (coactosin A) promoter, both of which are active under axenic conditions and in cells grown on bacteria. Requirements for the expression of reporter constructs as well as knock-in and knock-out constructs make it necessary to use our plasmid repertoire, but unfortunately limits the use of vectors already created that use the inefficient act6 promoter.
Promoter efficiencies are especially critical for transfections that depend on a single integration event into the correct genomic locus. Due to our improvement of promoters driving the resistance gene expression, enough resistance protein is produced from single locus integrations. A major concern was the favoring of multiple integrations into the genome when using hygromycin or G418 as described. No favoring of multiple integrations has been observed so far, as reported previously for G418 selections using older promoter systems25. This means that both hygromycin and G418 are suitable selectable markers for the generation of clean knock-outs and knock-ins. Unfortunately, the selectable marker blasticidin does not work under non-axenic conditions. This is a major disadvantage of our method, since the blasticidin resistance cassette is routinely used to generate knock-out constructs in D. discoideum. Constructs that were already generated with blasticidin resistance cassettes will need to be rederived using one of the workable selectable markers. Another possibility to overcome this limitation is to combine the recently established axenic D. discoideum CRISPR technology with this transfection protocol26. The generation of suitable, single guide RNAs (sgRNAs) is simpler and faster than the reconstruction of complete knock-out constructs. For future directions, the possibility of generating multiple knock-outs using CRISPR/Cas9 combined with this transfection protocol is appealing and may pave the way for many researchers in the Dictyostelium community. However, the workability of the established transient expression system used for CRISPR/Cas9 in axenic grown cells should be carefully examined.
The act5 knock-in system presented offers a reliable and safe integration system for the generation of stable cell lines in D. discoideum, with the advantage of similar sites established in other organisms22. It also depends on a single integration event in the genome and offers many possibilities for different research directions. The act5 promoter is strongly active and guarantees almost homogenous expression27 independent of the cultivation conditions. Fluorescent reporter proteins can easily be integrated into this safe haven locus using the engineered targeting plasmids. This can be useful, for example, for cell-tracking purposes, as shown here in a mix experiment. The cells show minimal cell-to-cell expression variability, which can assist in automated cell tracking. Importantly, the insertion of a desired sequence into the act5 locus appears phenotypically neutral28,29. As the expression does not depend on a selectable marker to maintain protein expression, as seen in random integrants or extrachromosomal vector-bearing cell lines, the act5 system can be useful for rescue experiments, as well. Since the cell lines are homogenous, all cells can be analyzed. This is not possible using an extrachromosomal plasmid for expression, in which there is considerable heterogeneity in expression.
In addition to these general improvements for all strains, the ability to use non-axenic wild-type cells for molecular research allows an assessment of the effects of accumulated mutations in current laboratory strains. Multiple genetic rearrangements has been found since the adoption of the Ax2 strain in 1970, as shown by previously published microarray analyses26. Synthetic phenotypes are observed because of the presence of these mutations. For example, a reported phg2 mutant shows decreased adhesion in one laboratory strain background and increased adhesion in another3. Such conflicting data can now be resolved by repeating the experiment in the common ancestor strain (in this case, DdB). The strength of this approach has been recently shown for the small GTPase RasS30,31.
The ability to perform genetic experiments in any D. discoideum strain expands the potential of new research directions that depend on the use of wild isolates, notably those involving social evolution, kin recognition, and the sexual cycle22.

The Dictyostelium genus are soil-living social amoeba that mainly feed on bacteria. Being placed in the phylum Amoebozoa, a large number of species have been isolated that can be grouped into four different clades1. The species Dictyostelium discoideum (D. discoideum) has become a popular model organism to study complex cellular processes such as cell migration and phagocytosis. To control and standardize experimental conditions, axenic cell lines have been developed that are able to grow in complex or defined liquid medium in the absence of bacteria2. Of particular importance are the Ax2, Ax3, and Ax4 strains, which were all generated in the 1970s and ultimately derived from a single wild isolate NC43. Tools for genetic engineering were developed in these axenic strains, resulting in the first published knockout in 19874,5. Protocols were further developed and optimized for use under axenic conditions6,7.
Adaptation of these protocols to wild-type D. discoideum strains that are not able to grow in liquid broth have been attempted by several laboratories. However, this has not become fully successful since the transfection protocols are complex and lack efficiency, in part due to the capacity of the bacteria to act as a sink for the selective reagents8,9. As a result, essentially all molecular data on D. discoideum comes from descendants of a single wild-type isolate. We wanted to overcome this limitation and develop a method to genetically modify D. discoideum cells independent of their ability to grow in liquid medium. The need for such a method can be explained by the observation that it was assumed in the past that the mutations allowing axenic growth were mainly neutral and did not impair cell physiology. This supposition is only partially correct. In general, there are two notable differences; first, between the different isolated axenic strains, and second, when these axenic strains are compared with non-axenic wild isolates8,9.
Perhaps the most critical factor is the key axenic gene, axeB, that was identified recently as RasGAP NF1. The major function of NF1 as a RasGAP is to restrain Ras activity3. The deletion of the enzyme in all axenic strains leads to excessive Ras activity manifested as the formation of large active Ras patches. These enlarged Ras patches lead to the accumulation of PIP3 in the plasma membrane. Those coincidental appearing patches of PIP3 and active Ras are a template for the formation of a circular ruffle that eventually closes and leads to the formation of macropinosomes10. The consequence is an excessive increase in macropinocytic activity. Macropinocytosis is an actin-driven process. A competition for cytoskeletal components for the formation of either macropinosomes or pseudopods is the result. Its impact on cell behaviour is reflected in the almost complete prevention of chemotaxis of vegetative cells to folate11. The massively enlarged PIP3 patches are very persistent. Even in starved cells, the PIP3 patches remain and can be misinterpreted as pseudopods, which can cause problems interpreting studies on chemotaxis to cAMP.
In some cases, the NF1 mutation is experimentally useful. This leads us to a second motivation for developing a transfection method for bacterially grown D. discoideum cells, since the increase in macropinocytosis rate makes axenic cells valuable for investigating fundamental aspects of this process12. However, mutation in the genes required for macropinocytosis, such as Ras and PI3-kinases10, have almost abolished axenic growth, making it necessary to manipulate these cells through growth on bacteria. Another reason that renders bacteria-based transfections valuable is the increasing use of Dictyostelids to explore questions in the evolution of multi-cellularity13,14, kin recognition15,16, and altruistic cellular behaviour, which mainly depend on the use of freshly isolated wild-type isolates17. All mentioned research areas can be facilitated by efficient methods for genetic manipulation of wild isolates, which are non-axenic and do not grow in liquid broth.
Our protocols allow for overcoming of the described limitations. Taken together, the possibility of performing genetic manipulations with bacterial grown D. discoideum cells holds benefits for all Dictyostelium researchers, even if it is just the increased speed of the selection process due to faster growth of the amoeba (4 h doubling time) on bacteria compared to growth in axenic media (10 h doubling time).

<table border="0" cellpadding="0" cellspacing="0" style="width:820px;" width="821">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Equipment</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Eppendorf Microcentrifuges 5424/5424R</td>
			<td style="width:184px;">ThermoScientific</td>
			<td style="width:119px;">05-400-002</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:351px;">Eppendorf 5702R Centrifuges with A-4-38 Model Rotor</td>
			<td style="width:184px;">ThermoScientific</td>
			<td style="width:119px;">12823252</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Eppendorf Mastercycler Nexus Thermal Cyclers</td>
			<td style="width:184px;">Sigma Aldrich</td>
			<td style="width:119px;">EP6331000025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Gene Pulser X Cell, including CE &#38; PC modules</td>
			<td style="width:184px;">BioRad</td>
			<td style="width:119px;">1652660</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Safety Cabinet Foruna - SCANLAF</td>
			<td style="width:184px;">Labogene</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">BioPhotometer Plus</td>
			<td style="width:184px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">CLSM 710</td>
			<td style="width:184px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Media &#38; Agaroses</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">LoFlo Medium</td>
			<td style="width:184px;">Formedium</td>
			<td style="width:119px;">LF0501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Seakem GTG Agarose</td>
			<td style="width:184px;">Lonza</td>
			<td style="width:119px;">50071</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Low EEO Agarose</td>
			<td style="width:184px;">BioGene</td>
			<td style="width:119px;">300-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Purified Agar</td>
			<td style="width:184px;">Oxoid</td>
			<td style="width:119px;">LP0028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">SM broth</td>
			<td style="width:184px;">Formedium</td>
			<td style="width:119px;">SMB0102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">2x LB broth</td>
			<td style="width:184px;">Formedium</td>
			<td style="width:119px;">LBD0102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Chemicals</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">SYBR Safe DNA Gel Stain</td>
			<td style="width:184px;">ThermoScientific</td>
			<td style="width:119px;">S33102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">1 Kb Plus DNA Ladder</td>
			<td style="width:184px;">ThermoScientific</td>
			<td style="width:119px;">10787018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">1 Kb DNA Ladder</td>
			<td style="width:184px;">NEB</td>
			<td style="width:119px;">N3232L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">HEPES free acid</td>
			<td style="width:184px;">Sigma Aldrich/Merck</td>
			<td style="width:119px;">391340 EMD</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">KH2PO4</td>
			<td style="width:184px;">VWR</td>
			<td style="width:119px;">P/4800/53</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">Na2HPO4 * 2 H2O</td>
			<td style="width:184px;">VWR</td>
			<td style="width:119px;">10028-24-7</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">MgCl2 * 6 H2O</td>
			<td style="width:184px;">Sigma Aldrich/Merck</td>
			<td style="width:119px;">5982 EMD</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:351px;">CaCl2 * 2 H2O</td>
			<td style="width:184px;">Sigma Aldrich</td>
			<td style="width:119px;">442909</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Folic Acid</td>
			<td style="width:184px;">Sigma Aldrich</td>
			<td style="width:119px;">F7876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">KOH</td>
			<td style="width:184px;">VWR</td>
			<td style="width:119px;">26668.263</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Cultur Dishes</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:351px;">96 Well Cell Culture Cluster, Flat Bottom with Low Evaporation Lid, Tissue Culture Treated</td>
			<td style="width:184px;">Corning Incorporated</td>
			<td style="width:119px;">3595</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:351px;">6 Well Cell Culture Cluster, Flat Bottom with Lid, Tissue Culture Treated</td>
			<td style="width:184px;">Corning Incorporated</td>
			<td style="width:119px;">3516</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:351px;">100 x 20 mm style Tissue Culture Dish, Tissue Culture Treated</td>
			<td style="width:184px;">Corning Incorporated</td>
			<td style="width:119px;">353003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mm Glass Bottom Microwell Dishes No1.5</td>
			<td style="width:184px;">MatTek Corporation</td>
			<td style="width:119px;">P50G-1.5-30-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Antibiotics</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:351px;">Geneticin G418-sulphate</td>
			<td style="width:184px;">Gibco by Life Technologies</td>
			<td style="width:119px;">11811-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Hygromycin B Gold</td>
			<td style="width:184px;">InvivoGen</td>
			<td style="width:119px;">ant-hg-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Additional Consumables</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">2mm gap electroporation cuvettes, long electrode</td>
			<td style="width:184px;">Geneflow Limited</td>
			<td style="width:119px;">E6-0062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">10 &#181;l Inoculating Loop, Blue 10 Micro L</td>
			<td style="width:184px;">ThermoScientific</td>
			<td style="width:119px;">129399</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Spreader, L-shaped, sterile</td>
			<td style="width:184px;">greiner bio-one</td>
			<td style="width:119px;">730190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Combitips advanced 5 ml</td>
			<td style="width:184px;">Eppendorf BIOPUR</td>
			<td style="width:119px;">30089669</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Kits</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">ZR Plasmid Miniprep Classic</td>
			<td style="width:184px;">Zymoresearch</td>
			<td style="width:119px;">D4016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Quick DNA Miniprep Kit</td>
			<td style="width:184px;">Zymoresearch</td>
			<td style="width:119px;">D3025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Zymoclean DNA Recovery Kit</td>
			<td style="width:184px;">Zymoresearch</td>
			<td style="width:119px;">D40002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Enzymes</td>
			<td style="width:184px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Restriction enzymes</td>
			<td style="width:184px;">NEB</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">OneTaq 2X Master Mix with Standard Buffer</td>
			<td style="width:184px;">NEB</td>
			<td style="width:119px;">M0482L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">T4 DNA Ligase</td>
			<td style="width:184px;">NEB</td>
			<td style="width:119px;">M0202S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">PrimeSTAR Max DNA Polymerase</td>
			<td style="width:184px;">TakaraBio</td>
			<td style="width:119px;">R045A</td>
			<td></td>
		</tr>
	</tbody>
</table>

genetic engineering of dictyostelium discoideum cells based on selection and growth on bacteria

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.

Extrachromosomal plasmids are used for reporter studies, which aim to identify the localization of certain proteins inside a cell or changes in cellular structure of mutant cells. For many approaches, such as monitoring of the cell cycle, it is crucial to express two reporters at the same time. This is now possible using our dual reporter extrachromosomal plasmid system (Table 1). On day 1, cells were transfected before adding the selectable marker G418 after 5 h (Figure 1). In the example, NC4, DdB, Ax2, and the independently derived wild isolates V12M2 and WS2162 (Supplementary Table 1) were transfected with the plasmid pPI289, which encodes for GFP-TubulinA, a marker for microtubules and mCherry-PCNA, a protein that is used for monitoring the cell cycle (Figure 2A). After 32 h, the cells were observed under the microscope. The majority of cells expressed both fluorescent-labeled fusion proteins, consistent with previous reports that expression of two reporters from the same plasmid shows similar expression levels, which is nearly impossible when using two different plasmids18,19. A representative cell for each cell line (NC4, DdB, Ax2, V12M2, and WS2162) expressing the desired dual reporter is shown in Figure 2B. The transfection efficiencies are summarized in Figure2C. NC4-derived cell lines show the best transfection efficiencies. However, for cell lines V12M2 and WS2162, a considerably high number of transfectants was obtained.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58981/58981fig2.jpg" /> 
Figure 2: Expression of an extrachromosomal plasmid. (A) Extrachromosomal plasmids are directly transfected in circular form. As an example, the dual reporter pPI289 is shown. The NgoMIV sites indicate insertion of the second reporter into the extrachromosomal expression plasmid. (B) Z-projection of a representative cell expressing GFP-TubulinA (cytoplasmic) and mCherry-PCNA (largely nuclear) for five different wild-type cell lines used (NC4, DdB, Ax2, V12M2, WS2162). (C) The transfection efficiencies for the five cell lines pictured in (B) were calculated. Shown is the mean of two experiments. Error bars indicate &#177; SD. <a href="https://www.jove.com/files/ftp_upload/58981/58981fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Integration of a targeting vector into a specified genomic locus is more challenging and requires more careful analysis of the generated cell line. In Figure 3, an act5-mCherry KI in NC4 is attempted. First, the plasmid must be linearized to increase the frequency of recombination events following transfection. For this, the plasmid pDM1514 is cut with NgoMIV. Two bands are obtained after running the digest on an agarose gel. The 4127 bp band contains the desired construct (Figure 3). For transfection, the digested DNA must be extracted from the gel and purified using a gel extraction kit following the manufacturer&#39;s instructions.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58981/58981fig3.jpg" /> 
Figure 3: Preparation of act5 knock-in and DNA for transfection. An example of the use of an act5 knock-in the plasmid pDM1514 is shown. The steps for preparation are listed as follows. (A) Before electroporation, linearize the plasmid using the indicated NgoMIV sites. (B, C) Run the cut plasmid on an agarose gel until the two expected bands are properly separated. Cut out the 4127 bp band containing the recombination arms, mCherry, and the resistance-cassette, and gel extract the DNA. The DNA is now ready for transfection. <a href="https://www.jove.com/files/ftp_upload/58981/58981fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The purified DNA was used for transfection of NC4 cells. After 5-6 days of selection, clones were obtained. Representative transfection efficiencies and the amount of positive identified clones for several act5 knock-in attempts are summarized in Table 3. Two clones of the NC4 transfection were randomly chosen and analyzed by PCR (Figure 4A), and both showed the predicted band patterns expected of a knock-in and were further validated with Southern blot analysis to ensure a single integration event of the construct into the genome20. The blot shows a clear single integration in the desired act5 locus (Figure 4B). The generated NC4::act5-mCherry cell line can now be used in experiments.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58981/58981fig4.jpg" /> 
Figure 4: Validation of act5-mCherry KIs in NC4. (A) Scheme and control PCRs for the validation of positive integrations into act5 locus. The indicated primers were used to analyze two independent clones and the parent. Both clones show the expected bands for the resistance-cassette and downstream (P1) or upstream primer (P2), which are not present in the parental strain. The primer combination (P1/P2) confirms the correct integration of mCherry and resistance cassette into the act5 locus. The wild-type NC4 shows the expected 2800 bp band, while both KI clones lack this band and instead display a PCR product about 1400 base pairs larger. (B) Scheme for the used restriction digest and Southern blot. Both knock-in clones show the smaller 3400 bp band, which is the result of integration of the construct into the act5 locus, specifically the additional BclI site in the hygromycin resistance cassette. The wild-type control shows the expected 5.8 kb resulting from the two downstream located BclI sites. The blot was cropped and spliced for clarity. <a href="https://www.jove.com/files/ftp_upload/58981/58981fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>targeted construct into the act5 locus</td>
			<td>plasmid name</td>
			<td>number of occupied wells</td>
			<td>number of checked clones</td>
			<td>Positive clones</td>
			<td>Correct clones (%)</td>
			<td>Dictyostelium strain used</td>
			<td>transfection efficiency (transfectants/ 2x10^6/1 &#181;g DNA)</td>
		</tr>
		<tr>
			<td>LifeAct-mCherry</td>
			<td>pPI226</td>
			<td>7</td>
			<td>7</td>
			<td>1</td>
			<td>14.2</td>
			<td>AX2</td>
			<td>3.5x10^-6</td>
		</tr>
		<tr>
			<td>LifeAct-GFP</td>
			<td>pPI227</td>
			<td>12</td>
			<td>12</td>
			<td>5</td>
			<td>41.6</td>
			<td>AX2</td>
			<td>6x10^-6</td>
		</tr>
		<tr>
			<td>GFP</td>
			<td>pDM1513</td>
			<td>3</td>
			<td>3</td>
			<td>2</td>
			<td>66.6</td>
			<td>AX2</td>
			<td>1.5x10^-6</td>
		</tr>
		<tr>
			<td>mCherry</td>
			<td>pDM1514</td>
			<td>3</td>
			<td>3</td>
			<td>1</td>
			<td>33.3</td>
			<td>AX2</td>
			<td>1.5x10^-6</td>
		</tr>
		<tr>
			<td>GFP</td>
			<td>pDM1513</td>
			<td>66</td>
			<td>9</td>
			<td>5</td>
			<td>55.5</td>
			<td>AX2</td>
			<td>3.3x10^-5</td>
		</tr>
		<tr>
			<td>mCherry</td>
			<td>pDM1514</td>
			<td>221</td>
			<td>12</td>
			<td>10</td>
			<td>83.3</td>
			<td>AX2</td>
			<td>1.1x10^-4</td>
		</tr>
		<tr>
			<td>H2B-mCherry</td>
			<td>pPI420</td>
			<td>3</td>
			<td>3</td>
			<td>1</td>
			<td>33.3</td>
			<td>AX2</td>
			<td>1.5x10^-6</td>
		</tr>
		<tr>
			<td>H2B-mCherry</td>
			<td>pPI420</td>
			<td>7</td>
			<td>7</td>
			<td>6</td>
			<td>85.7</td>
			<td>AX2</td>
			<td>3.5x10^-6</td>
		</tr>
		<tr>
			<td>mCherry</td>
			<td>pDM1514</td>
			<td>10</td>
			<td>10</td>
			<td>7</td>
			<td>70</td>
			<td>DdB</td>
			<td>5x10^-6</td>
		</tr>
		<tr>
			<td>mCherry</td>
			<td>pDM1514</td>
			<td>240</td>
			<td>12</td>
			<td>11</td>
			<td>91.6</td>
			<td>DdB</td>
			<td>1.2x10^-4</td>
		</tr>
		<tr>
			<td>mCherry</td>
			<td>pDM1514</td>
			<td>320</td>
			<td>12</td>
			<td>12</td>
			<td>100</td>
			<td>NC4</td>
			<td>1.6x10^-4</td>
		</tr>
	</tbody>
</table>
Table 3: Transfection efficiencies and the amount of obtained positive transfectants for the generation of act5 KIs in different strain backgrounds22.
The act5 locus offers relatively homogenous expression of the integrated reporter21. The generated NC4::act5-mCherry cells allow mix experiments to be performed with other act5 knock-ins using a different fluorescent protein such as GFP. To emphasize the great advantage of this system, mixing experiments with Ax2::act5-GFP are shown. Due to the inability to transfect non-axenic wild-type cells, this type of approach could not be performed before. Mix experiments are an important tool to analyze cell behavior, because they allow a direct comparison between different cell lines experiencing identical experimental conditions. NC4 cells grow faster on bacterial lawns than Ax2 cells (Figure 5A). This may be due to a higher capacity to phagocytose bacteria or improved ability to move and show chemotaxis towards a food source. Using an under-agar folate chemotaxis assay, direct comparison of a population of NC4::act5-mCherry and Ax2::act5-GFP was performed, showing that NC4::act5-mCherry are much more efficient in folate sensing. After 4 h, more NC4::act5-mCherry cells were able to crawl under the agarose than Ax2::act5-GFP cells (Figure 5B). Analyzing standard metrics of chemotaxis for the cells migrating under the agarose revealed that NC4::act5-mCherry cells were faster and showed stronger chemotactic response than Ax2::act5-GFP cells (Figure 5C-E).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58981/58981fig5.jpg" /> 
Figure 5: Using act5 KIs for image-based chemotaxis mix experiments. (A) NC4::act5-mCherry and Ax2::act5-GFP expressing the indicated fluorescent protein from the act5 locus were analyzed for the ability to grow on a bacterial lawn. After 4 days, the plaque diameter arisen from solitary plated Dictyostelium cells were measured. Non-axenic act5::NC4 cells make significantly bigger plaques than axenic Ax2::act5-GFP cells (mean &#177; SD, ***p &#60; 0.0001, n = 3, scale bar 5 mm). (B) For use of the under-agarose folate chemotaxis assay22 to directly compare the chemotactic abilities of the NC4::act5-mCherry and Ax2::act5-GFP used in (A), bacteria- grown amoebae of both strains were mixed in a 50:50 ratio. Cells were allowed to crawl under the agarose. After 4 h, cells that were migrating up the folate gradient were imaged using a confocal microscope. The number of NC4::act5-mCherry and Ax2::act5-GFP cells was then determined. NC4::act5-mCherry cells were more efficient in sensing folate. About 10-fold more NC4::act5-mCherry cells were found compared to Ax2::act5-GFP cells (mean &#177; SD, ***p &#60; 0.0001, n = 6, scale bar 100 &#181;m). (C, D) The cells were filmed for 60 min, and their speed and chemotactic index were calculated. After pre-selection of the most chemotactically responsive cells (only those that migrated under the agarose), Ax2::act5-GFP cells showed lower values for both cell speed and chemotaxis. Fifty cells per cell line were analyzed. (median &#177; SD, *p &#60; 0.01, n = 3). (E) The tracks of NC4::act5-mCherry and Ax2::act5-GFPact5 knock-ins are plotted over 60 min, showing the more directed movement towards the chemoattractent source of the NC4::act5-mCherry cells (median &#177; SD, ***p &#60; 0.0001, n = 6). <a href="https://www.jove.com/files/ftp_upload/58981/58981fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The act5 knock-in cell lines can also be used for flow cytometry. As with growth on bacteria, there are major differences in development between axenic strains and non-axenic wild-types. After development, the fruiting bodies of NC4 cells are approximately twice as big as those derived from Ax2 cells. If cells are mixed, an intermediate sized fruiting body is obtained. To analyze the contribution of the both cell lines more quantitatively, flow cytometry was used. These analyses showed clearly that the intermediate-sized fruiting bodies are due to different levels of contribution from both cell lines. While NC4::act5-mCherry cells made up about 75% of the measured spores, Ax2::act5-GFP contributed only 25%, revealing a potential fitness advantage for non-axenic strains (Figure 6). Since the analysis does not monitor the stalk cell population, there are two possibilities to explain the imbalance in development between Ax2 and NC4. One possibility is that Ax2 cells contribute mainly to the stalk cell population, rather than entering the spore cell population. Alternatively, more NC4 cells may enter the developmental cycle, with Ax2 relatively delayed, and they are consequently unable to contribute to the assembly of a fruiting body. The possibility to transfect non-axenic wild-type cells further develops other approaches and greatly simplifies experimental procedures.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58981/58981fig6.jpg" /> 
Figure 6: Act5 KIs allow analysis of mix experiments using flow cytometry. (A) NC4::act5-mCherry and Ax2::act5-GFP were developed separately or in a 50:50 mixture on non-nutrient agar plates. NC4::act5-mCherry cells form larger fruiting bodies than Ax2::act5-GFP. The mix instead shows an intermediate size (scale bar 5 mm). Confocal fluorescence light microscopy suggests a higher amount of NC4::act5-mCherry spores in the spore heads derived from mixes. (B) To quantify this observation, the amounts of spores in harvested spore heads from the mixing experiment in (A) were analyzed by flow cytometry. About 75% of the spores originated from NC4::act5-mCherry cells, with only 25% from Ax2::act5-GFP cells. (C) Representative distribution of spores from both cell lines shown in a flow cytometry scatter. About 0.05% show positive mCherry and GFP signals, suggesting that parasexual fusion processes have occurred or that spores are sticking to each other. <a href="https://www.jove.com/files/ftp_upload/58981/58981fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Cell line</td>
			<td>Genetically background</td>
			<td>Reference number</td>
			<td>Published before</td>
			<td>Type</td>
		</tr>
		<tr>
			<td>AX2 (Ka)</td>
			<td></td>
			<td>DBS0235521</td>
			<td>Bloomfield et al., 2008</td>
			<td>wild type</td>
		</tr>
		<tr>
			<td>NC4 (S)</td>
			<td></td>
			<td></td>
			<td>Bloomfield et al., 2008</td>
			<td>wild type</td>
		</tr>
		<tr>
			<td>act5::mCherry Clone 5</td>
			<td>NC4</td>
			<td>HM1912</td>
			<td>Paschke et al., 2018</td>
			<td>act5 knock-in</td>
		</tr>
		<tr>
			<td>act5::GFP Clone 2</td>
			<td>AX2</td>
			<td>HM1930</td>
			<td>Paschke et al., 2018</td>
			<td>act5 knock-in</td>
		</tr>
		<tr>
			<td>V12M2</td>
			<td></td>
			<td></td>
			<td>Bloomfield et al., 2008</td>
			<td>wild type</td>
		</tr>
		<tr>
			<td>WS2162</td>
			<td></td>
			<td></td>
			<td>Bloomfield et al., 2008</td>
			<td>wild type</td>
		</tr>
	</tbody>
</table>
Supplementary Table 1: Different strains of Dictyostelium studied.

Watch this Scientific Journal Video about Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria at JoVE.com

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

キイロタマホコリカビの選択に基づいて細胞と細菌の成長の遺伝子工学

Dictyostelium discoideum is a popular model organism to study complex cellular processes such as cell migration, endocytosis, and development. The utility of the organism is dependent on the feasibility of genetic manipulation. Here, we present methods to transfect Dictyostelium discoideum cells that overcome existing limitations of culturing cells in liquid media.

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...

genetic-engineering-dictyostelium-discoideum-cells-based-on-selection

Research

JoVE Journal

Genetics

12.7K ビュー.  MRC Laboratory of Molecular Biology. このプロトコルの価値は、その株が液体培地で無比に成長するかどうかに関係なく、ジクチオステリウム・ディスコジウムのほぼすべての株を分子遺伝学によって操作することができるということです。この手法のもう 1 つの利点は、高速で簡単なことです。ジクチオステリウムを混合し始める前に、エキソコモソームプラスミドを用いてトランスフェクト細胞に移調?...

ビデオ: Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Genetic Engineering of an Unconventional Yeast for Renewable Biofuel and Biochemical Production

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic characteristics, this unconventional yeast is not only a good model for the study of the fundamental nature of fungal differentiation but is also a promising microbial platform for biochemical production and various biotechnological applications, which require extensive genetic manipulations. However, genetic manipulations of Y. lipolytica have been limited due to the lack of an efficient and stable genetic transformation system as well as very high rates of non-homologous recombination that can be mainly attributed to the KU70 gene. Here, we report an easy and rapid protocol for the efficient genetic transformation and for gene deletion in Y. lipolytica Po1g. First, a protocol for the efficient transformation of exogenous DNA into Y. lipolytica Po1g was established. Second, to achieve the enhanced double-crossover homologous recombination rate for further deletion of target genes, the KU70 gene was deleted by transforming a disruption cassette carrying 1 kb homology arms. Third, to demonstrate the enhanced gene deletion efficiency after deletion of the KU70 gene, we individually deleted 11 target genes encoding alcohol dehydrogenase and alcohol oxidase using the same procedures on the KU70 knockout platform strain. It was observed that the rate of precise homologous recombination increased substantially from less than 0.5% for deletion of the KU70 gene in Po1g to 33%-71% for the single gene deletion of the 11 target genes in Po1g KU70&#916;. A replicative plasmid carrying the hygromycin B resistance marker and the Cre/LoxP system was constructed, and the selection marker gene in the yeast knockout strains was eventually removed by expression of Cre recombinase to facilitate multiple rounds of targeted genetic manipulations. The resulting single-gene deletion mutants have potential applications in biofuel and biochemical production.

We herein report methods on the molecular genetic manipulation of the Yarrowia lipolytica Po1g strain for improved gene deletion efficiency. The resulting engineered Y. lipolytica strains have potential applications in biofuel and biochemical production.

再生可能バイオ燃料および生化学的生産のための独創的な酵母の遺伝子工学

Yarrowia lipolytica is a non-pathogenic, dimorphic and strictly aerobic yeast species. Owing to its distinctive physiological features and metabolic ...

遺伝学

Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial culture device that continuously monitors bacterial growth and dynamically adjusts the drug concentration to constantly challenge the bacteria as they evolve to acquire drug resistance. The device features a working volume of ~10 ml and is fully automated and equipped with optical density measurement and micro-pumps for medium and drug delivery. To validate the platform, we measured the stepwise acquisition of trimethoprim resistance in Escherichia coli MG 1655, and integrated the device with a multiplexed microfluidic platform to investigate cell morphology and antibiotic susceptibility. The approach can be up-scaled to laboratory studies of antibiotic drug resistance, and is extendible to adaptive evolution for strain improvements in metabolic engineering and other bacterial culture experiments.

We describe a low cost, configurable morbidostat that enables the characterization of antibiotic drug resistance by dynamically adjusting the drug concentration. The device can be integrated with a multiplexed microfluidic platform. The approach can be scaled up for laboratory antibiotic drug resistance studies.

設計と低コストの使用、自動Morbidostat細菌の適応進化のための抗生物質選択下

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial ...

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

細菌接合に関与する転写タンパク質の配列特異的分析のための接合交配アッセイ

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species.
Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family,&#160;it could be easily used to other prokaryote and eukaryote species

Here, we present a protocol for tracing genomic DNA (gDNA) contamination in RNA samples. The presented method utilizes primers specific for the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) genes. The method is suited for reliable and sensitive detection of DNA contamination in most eukaryotes and prokaryotes.

リボソーム DNA に基づいて RNA サンプルの DNA 汚染の評価

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time ...

Single Molecule Analysis of Laser Localized Psoralen Adducts

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies. DNA fibers are often used to display the progress of replication forks by immunofluorescence of nucleoside analogues incorporated during short pulses. Immunoquantum dots have been widely employed for single molecule imaging. In the new approach, DNA fibers from cells carrying laser localized ICLs are spread onto microscope slides. The tagged ICLs are displayed with immunoquantum dots and the inter-lesion distances determined. Replication fork collisions with ICLs can be visualized and different encounter patterns identified and quantitated.

Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

レーザーローカライズソラレン付加物の単一分子解析

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live ...

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

哺乳類細胞におけるCRISPR / Cas9媒介遺伝子ノックアウトの選択依存性および独立した世代

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

分離の特性と人間の歯小嚢細胞のマイクロ Rna を用いた遺伝子組み換え

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame (ORF) annotation impose two arbitrary criteria: a minimum length of 100 codons and a single ORF per transcript. However, a growing number of studies report expression of proteins from allegedly non-coding regions, challenging the accuracy of current genome annotations. These novel proteins were found encoded either within non-coding RNAs, 5' or 3' untranslated regions (UTRs) of mRNAs, or overlapping a known coding sequence (CDS) in an alternative ORF. OpenProt is the first database that enforces a polycistronic model for eukaryotic genomes, allowing annotation of multiple ORFs per transcript. OpenProt is freely accessible and offers custom downloads of protein sequences across 10 species. Using OpenProt database for proteomic experiments enables novel proteins discovery and highlights the polycistronic nature of eukaryotic genes. The size of OpenProt database (all predicted proteins) is substantial and need be taken in account for the analysis. However, with appropriate false discovery rate (FDR) settings or the use of a restricted OpenProt database, users will gain a more realistic view of the proteomic landscape. Overall, OpenProt is a freely available tool that will foster proteomic discoveries.

OpenProt is a freely accessible database that enforces a polycistronic model of eukaryotic genomes. Here, we present a protocol for the use of OpenProt databases when interrogating mass spectrometry datasets. Using OpenProt database for analysis of proteomic experiments allows for discovery of novel and previously undetectable proteins.

非正規の開いたリーディング ・ フレームから翻訳された蛋白質を発表する OpenProt データベースを利用した質量分析を用いたプロテオミクス解析

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.

CRISPR-Cas9 technology provides an efficient method to precisely edit the mammalian genome in any cell type and represents a novel means to perform genome-wide genetic screens. A detailed protocol discussing the steps required for the successful performance of pooled genome-wide CRISPR-Cas9 screens is provided here.

哺乳類細胞におけるCRISPRベースの遺伝子スクリーンのプール

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian ...

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

ゲノムワイド往復半角化分析によるサッカロマイセス酵母の種間の熱耐性の遺伝学的マッピング

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...