These studies were partially funded by a grant from the George F. Haddix Fund of Creighton University (KMD). Figure 1 is generated in biorender.com, and Figure 3 is generated in benchling.com.

The details of the species, reagents, and equipment used in this study are listed in the Table of Materials. The sequence of the 17,004 base pair pGRUB construct is provided in Supplementary File 1.
1. Culturing trophozoites
<ol>
	<li>Thaw frozen N. gruberi trophozoites at 37 &#176;C for 3 min.</li>
	<li>Inoculate trophozoites into T25 flasks in modified peptone/yeast extract/nucleic acid/folic acid/hemin with 10% fetal calf serum (PYNFH) media plus 2% buffer (disodium phosphate, potassium dihydrogen phosphate).</li>
	<li>Culture the trophozoites at 25&#160;&#176;C until ~80% confluent, where they exhibit an amoeboid morphology (Figure 2A). 
	NOTE: When initially reviving a culture of trophozoites from their frozen state, it can take up to 5-7 days for a T25 to become confluent. Decant media every 3-4 days and replace with fresh media.</li>
	<li>Place the confluent flasks on ice for 5-10 min to release the adhered trophozoites from the plastic. Gently swirl the flask to dislodge any remaining adherent cells. The trophozoites now have a &#39;rounded up&#39; morphology (Figure 2B).</li>
	<li>Split the cultures 1:2 to 1:4 into new tissue culture flasks.</li>
	<li>Remove the PYNFH and replace it with sterile encystment media (120 mM sodium chloride, 0.03 mM magnesium chloride, 1 mM disodium phosphate, 1 mM potassium dihydrogen phosphate, 0.03 mM calcium chloride, 0.02 mM iron chloride, pH 6.8)22 to encyst the N. gruberi. 
	NOTE: The majority of trophozoites become encysted by 72 h (Figure 2C). Excystment should be completed within 72 h.</li>
	<li>Remove cysts from flasks, centrifuge at 600 x g for 10 min at 20&#160;&#176;C, and resuspend cysts in PYNFH media with 10% fetal calf serum to excyst the cells. Incubate at 25&#160;&#176;C for 24 h until trophozoites begin to emerge.</li>
</ol>
2. Transfecting trophozoites
<ol>
	<li>Plate the trophozoites into a 6-well flat-bottomed tissue culture plate (1 mL of 1 x 106 trophozoites/mL) and culture at 25&#160;&#176;C.</li>
	<li>Transfect the cells when they reach 70%-80% confluent (~10.4 x 104 cells/cm2 in 6-well clusters). Warm the transfection reagent to room temperature before use.</li>
	<li>Prepare the plasmid-transfection reagent mixture as follows: 75 ng of plasmid DNA and 0.225 &#181;L of the transfection reagent in 200 &#181;L of PYNFH media without FBS. Prepare sufficient plasmid-transfection reagent mixture to perform transfection in duplicate. 
	NOTE: A bacterial plasmid containing a full-length clone of N. gruberi CERE (pGRUB) was used in this study. Empty plasmid (pGEM) was used as a negative control (Figure 3).</li>
	<li>Incubate the plasmid-transfection reagent mixture at room temperature for 10 min.</li>
	<li>Remove ~800 &#181;L of medium from the trophozoite monolayers just prior to transfection.</li>
	<li>Put the plasmid-transfection reagent mixture on the trophozoites.</li>
	<li>Gently swirl the plate every 15 min to prevent the media from aggregating at the edges of the plate.</li>
	<li>Wash the monolayers once with 2 mL of growth medium after 1 h incubation at 25&#160;&#176;C.</li>
	<li>Treat the monolayers with 10 U DNase in 1 mL of 10x DNase buffer for 15 min at 37&#160;&#176;C to remove residual plasmid DNA, wash once with growth medium, add 2 mL of fresh media, and place at 25&#176;C. Incubate the plate for 24-36 h.</li>
	<li>Place the plate on ice for 10 min to release cells.</li>
	<li>Split one well (1:2) into two&#160;new wells.</li>
	<li>Collect trophozoites from the remaining wells for experimental purposes. 
	NOTE: All cultures are performed in duplicate. For each condition, 1 well is used for passage, and 1 is used for CERE isolation.</li>
</ol>
3. Isolating CERE from Naegleria
<ol>
	<li>Calculate the number of trophozoites in each well used for CERE isolation by trypan blue exclusion staining and a hemocytometer23.</li>
	<li>Place the trophozoites in a 2.0 mL tube.</li>
	<li>Wash trophozoites once in 100 mM sodium chloride (pH 7.5) via centrifugation (600 x g, 10 min, 4 &#176;C). 
	NOTE: Sodium chloride prevents the trophozoites from adhering to the sides of the tube.</li>
	<li>Remove the supernatant and resuspend cell pellets in 100 &#181;L of 100 mM EDTA (pH 7.5).</li>
	<li>Place the tube in a heat block for 15 min at 98&#160;&#176;C to lyse the cells and inactivate the enzymes in the trophozoites. 
	NOTE: This step inactivates DNases, which are abundant in Naegleria trophozoites. DNases may interfere with the ability to detect native CERE and the molecular clone in step 4. DNA yield is decreased when over ~3 x 106 cells are used in step 3.4.</li>
	<li>Centrifuge the tubes at top speed (16,000 x g) at 4&#160;&#176;C for 10 min to pellet the debris.</li>
	<li>Transfer the supernatants to a new tube.</li>
	<li>Ethanol precipitate the supernatants with 0.5 volumes of 7.5 M ammonium acetate (pH 7.5), 3 volumes of absolute ethanol, and 1 &#181;L of 10 mg/mL glycogen as a carrier.</li>
	<li>Invert the tube several times and place the tube at -80&#160;&#176;C for at least 1&#160;h or overnight.</li>
	<li>Warm the samples to room temperature.</li>
	<li>Centrifuge the tubes at room temperature for 10 min at 16,000 x g.</li>
	<li>Remove the supernatant and wash the pellet with 70% ethanol by centrifugation for 5 min at 16,000 x g (at room temperature).</li>
	<li>Remove ethanol and air-dry the pellet for 5 min.</li>
	<li>Resuspend the dried pellet in sterile deionized (DI) water. Normalize volume to 10,000 trophozoites per &#181;L.</li>
	<li>Store at -80&#160;&#176;C until use in PCR.</li>
</ol>
4. PCR detection of transformed trophozoites
<ol>
	<li>Use 20,000 cell equivalents of DNA, 600 nM primers (Table 1), and Taq polymerase and perform the PCR24. 
	NOTE: Primers that distinguish between native CERE and the cloned CERE are listed in Table 1, as are the PCR conditions. Figure 3 shows the location of the primers on the constructs. PCR will require optimization depending on the primers used as well as the specific PCR mix utilized.</li>
	<li>Microwave 0.8% agarose in 1x Tris base, acetic acid, and EDTA (TAE) buffer until the agarose is dissolved.</li>
	<li>Cool the agarose to ~50&#160;&#176;C at room temperature.</li>
	<li>Pour the solution into a gel casting tray containing a comb to form wells.</li>
	<li>Let the gel sit at room temperature until solidified.</li>
	<li>Place the gel in the electrophoresis apparatus.</li>
	<li>Decant 1x TAE into the electrophoresis chamber until the gel is covered.</li>
	<li>Remove the comb.</li>
	<li>Add 6x loading dye (0.25% bromphenol blue, 0.25% xylene cyanol, 30% glycerol) to samples and load the gel. Use a DNA ladder to determine the size of the PCR product.</li>
	<li>Attach the electrode, turn on the power supply, and run the gel at 80 V for ~1.5-2 h.</li>
	<li>Turn off the power and remove the gel.</li>
	<li>Stain the gel with DNA dye (e.g., 10 &#181;L of 10 mg/mL ethidium bromide) in 100 mL of DI water for 10 min.</li>
	<li>Destain the gel for 20 min in DI water.</li>
	<li>Visualize gel on an ultraviolet (UV) light system and document the results.</li>
</ol>

Using a CERE-Based Vector for Transfection of &lt;i&gt;Naegleria gruberi&lt;/i&gt; Trophozoites

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No financial conflicts of interest were declared.

The protocol outlined herein is very straightforward, although every construct will likely require some degree of optimization, particularly of the DNA-transfection reagent ratio, depending on the nature of the construct and the species of Naegleria used. We have only tested one commercially available transfection reagent using this protocol, but it is likely that several others may be effective. Given that a full-length clone of the CERE is used, containing a functional origin of replication and thus replicatin...

The Naegleria genus contains over 45 species, although it is unlikely that all members of the species have been identified1. Naegleria can exist in different forms: as trophozoites (amoebae), as flagellates, or, when resources are severely limited, as cysts1,2,3,4. The Naegleria genus is recognized for its one particularly dangerous species, Naegleria fowleri, known as &#39;the brain-eating amoeba&#39; (reviewed in1,2,3,4,5,6,7), which is the cause of the almost universally fatal primary amoebic meningoencephalitis.
Naegleria encode their rDNA on the CERE located in the nucleolus. Complete sequencing of three Naegleria species&#39; genomes confirms the absence of rDNA in the nuclear genome8,9,10,11. Based on the limited full-length CERE sequences, the CERE ranges from approximately 10 kbp to 18 kbp in length. CERE of each species of Naegleria carry a single rDNA cistron (containing the 5.8, 18, and 28S ribosomal DNA) on each of approximately 4,000 CERE per cell12,13,14. Other than rDNA, each CERE has a large non-rDNA sequence (NRS). CERE sizes vary between species; the differences are mainly due to the varying length of the NRS, as the rDNA sequences are highly conserved across the genus1,15,16,17,18,19,20. The mapping of a single origin of DNA replication within the N. gruberi CERE NRS21 provides strong support for the hypothesis that Naegleria CERE replicates independently of the nuclear genome.
N. gruberi is a non-pathogenic amoeba often used to study Naegleria biology. We developed a methodology for transforming N. gruberi trophozoites with a CERE clone from the same species to test the hypothesis that Naegleria amoebae tolerate and independently replicate CERE within the trophozoites. This was accomplished by transforming N. gruberi with a full-length clone of N. gruberi CERE into trophozoites and following the fates of the donor CERE clone by polymerase chain reaction (PCR). A general overview of the protocol is outlined in Figure 1. The data presented herein demonstrate that the donor clone can be detected through at least seven passages in tissue culture, as well as be maintained through encystment and excystment. These studies form the basis for a means to dissect how CERE replicates, as well as explore its use as a vector to transfect Naegleria.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Bio Rad</td>
			<td>161-3102</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Acetate</td>
			<td>Sigma Aldrich</td>
			<td>A-7330</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>C-4901</td>
			<td></td>
		</tr>
		<tr>
			<td>Crushed ice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Flasks, T-75</td>
			<td>Thermo Scientific</td>
			<td>130190</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Plate, 6-well</td>
			<td>Corning</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Sigma Aldrich</td>
			<td>D-4527</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA, 0.5 M</td>
			<td>Affymetrix</td>
			<td>15694</td>
			<td></td>
		</tr>
		<tr>
			<td>Electropheresis Gel Apparatus</td>
			<td>Amersham Biosciences</td>
			<td>80-6052-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Tubes, 1.5 mL</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Tubes, 2 mL</td>
			<td>Fisher Scientific</td>
			<td>05-408-138</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, 100%</td>
			<td>Decon Laboratories</td>
			<td>2716</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Sigma Aldrich</td>
			<td>E-8751</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>26140</td>
			<td></td>
		</tr>
		<tr>
			<td>Folic Acid</td>
			<td>Sigma Aldrich</td>
			<td>F7876-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneRuler 1 kb Plus Ladder</td>
			<td>Thermo Scientific</td>
			<td>SM1331</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>Fisher Scientific</td>
			<td>UN2789</td>
			<td></td>
		</tr>
		<tr>
			<td>GoTaq Green PCR Master Mix</td>
			<td>Promega</td>
			<td>M7122</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Block</td>
			<td>Thermo Scientific</td>
			<td>88871001</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Hausser Scientific</td>
			<td>1483</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemin</td>
			<td>Sigma Aldrich</td>
			<td>51280</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron Chloride</td>
			<td>Sigma Aldrich</td>
			<td>372870-256</td>
			<td></td>
		</tr>
		<tr>
			<td>Ligase</td>
			<td>NEB</td>
			<td>M2200S</td>
			<td></td>
		</tr>
		<tr>
			<td>Magneisum Chloride</td>
			<td>Fisher Scientific</td>
			<td>M33-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfuge</td>
			<td>Thermo Scientific</td>
			<td>MySpin 12</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon</td>
			<td>TMS</td>
			<td></td>
		</tr>
		<tr>
			<td>N. gruberi</td>
			<td>ATCC</td>
			<td>30224</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleic Acid</td>
			<td>Chem Impex Int&#8217;l</td>
			<td>#01625</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Gibco</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>pGEM</td>
			<td>Promega</td>
			<td>P2251</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate</td>
			<td>Sigma Aldrich</td>
			<td>P0662-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerPac HC Electropharesis Power Supply Unit</td>
			<td>Bio Rad</td>
			<td>1645052</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>MCB Reagents</td>
			<td>SX0420</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate, dibasic</td>
			<td>Sigma Aldrich</td>
			<td>S2554</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabletop Centrifuge</td>
			<td>eppendorf</td>
			<td>5415R</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris, base</td>
			<td>Sigma Aldrich</td>
			<td>T1503-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue, 0.4%</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>ViaFect Reagent</td>
			<td>Promega</td>
			<td>E4981</td>
			<td></td>
		</tr>
		<tr>
			<td>Weigh Scale</td>
			<td>Denver Instruments</td>
			<td>APX-60</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Gibco</td>
			<td>212750</td>
			<td></td>
		</tr>
	</tbody>
</table>

transfection of a molecular clone of naegleria gruberi rdna into n. gruberi trophozoites

All ribosomal genes of Naegleria trophozoites are maintained in a closed circular extrachromosomal ribosomal DNA (rDNA) containing element (CERE). While little is known about the CERE, a complete genome sequence analysis of three Naegleria species clearly demonstrates that there are no rDNA cistrons in the nuclear genome. Furthermore, a single DNA origin of replication has been mapped in the N. gruberi CERE, supporting the hypothesis that CERE replicates independently of the nuclear genome. This CERE characteristic suggests that it may be possible to use engineered CERE to introduce foreign proteins into Naegleria trophozoites. As the first step in exploring the use of a CERE as a vector in Naegleria, we developed a protocol to transfect N. gruberi with a molecular clone of the N. gruberi CERE cloned into pGEM7zf+ (pGRUB). Following transfection, pGRUB was readily detected in N. gruberi trophozoites for at least seven passages, as well as through encystment and excystment. As a control, trophozoites were transfected with the backbone vector, pGEM7zf+, without the N. gruberi sequences (pGEM). pGEM was not detected after the first passage following transfection into N. gruberi, indicating its inability to replicate in a eukaryotic organism. These studies describe a transfection protocol for Naegleria trophozoites and demonstrate that the bacterial plasmid sequence in pGRUB does not inhibit successful transfection and replication of the transfected CERE clone. Furthermore, this transfection protocol will be critical in understanding the minimal sequence of the CERE that drives its replication in trophozoites, as well as identifying regulatory regions in the non-ribosomal sequence (NRS).

Author Spotlight: Understanding the Propagation of Closed Circular Extrachromosomal rDNA Containing Element (CERE) in &lt;i&gt;Naegleria gruberi&lt;/i&gt;

Transfection of a Molecular Clone of Naegleria gruberi rDNA into N. gruberi Trophozoites

Our research represents the first step to understanding what sequence is contained in the closed circular extrachromosomal ribosomal DNA containing element, or CERE, are critical in permitting replication of the CERE. As well as understanding the DNA replication machinery of the CERE. This protocol addresses gaps in understanding how the CERE propagates a naegleria gruberi, and what components of the CERE are necessary for effective propagation.Our protocol offers a minimalistic method of transecting naegleria, aiming to reduce procedures that may disturb the cell. It thereby addresses how the CERE propagates in naegleria gruberi, and what components of the CERE are necessary for effective propagation. We have established that the species of naegleria that contain unique CERE that varies in both sequence length and nucleotide composition.We look to provide the groundwork for analyzing the molecular dynamics of the CERE for species within the naegleria genus. Our results have paved the way for questions regarding what components are necessary for CERE replication. And whether the ORI can be utilized for the propagation of foreign constructs in naegleria species.We aim to advance our research further by branching out into other species of the naegleria genus, and providing answers regarding CERE transfection at an intra-species level.

PCR of trophozoites that have been transfected with pGRUB demonstrates that the transfected CERE is detected through at least seven passages of the trophozoites, as well as encystment and excystment (Figure 4). The primers used in the PCR anneal to both the pGEM vector and the CERE sequence, thereby ensuring that the PCR does not detect native CERE. PCR following transfection of pGEM into trophozoites indicated that pGEM was negative (Figure 4), indicating that ...

This protocol describes a methodology to transfect Naegleria gruberi trophozoites with a construct that is maintained throughout passaging trophozoites in vitro, as well as through encystment and excystment.

All ribosomal genes of Naegleria trophozoites are maintained in a closed circular extrachromosomal ribosomal DNA (rDNA) containing element (CERE). While ...

transfection-molecular-clone-naegleria-gruberi-rdna-into-n-gruberi

Research

JoVE Journal

Biology

Culture and Transfection of Naegleria gruberi Trophozoites for Molecular Studies

To begin, place frozen Naegleria gruberi trophozoites at 37 degrees Celsius for three minutes. Culture the trophozoites in PYNFH media at 25 degrees Celsius until approximately 80%confluent. Place the confluent flask on ice for five to 10 minutes to release the adhered trophozoites from the plastic.Then gently swirl the flask to dislodge any remaining adherent cells. Replace the media with sterile encystment media to encyst the Naegleria gruberi cells. After 48 hours, remove the cysts from the flask.Centrifuge the cysts at 600 x g for 10 minutes at four degrees Celsius. After centrifugation, resuspend the cysts in two milliliters of PYNFH media with 10%fetal calf serum to excyst the cells. Incubate the cysts at 25 degrees Celsius for 72 hours until trophozoites emerge and reach 80%confluency.To transfect the trophozoites, first plate one milliliter of the trophozoites into a six-well, flat bottom tissue culture plate. Next, incubate the plasmid transfection reagent mixture at room temperature for 10 minutes. Pipette out approximately 800 microliters of medium from the trophozoites monolayers just prior to transfection.Then add 200 microliters of the plasmid transfection reagent mixture to the trophozoites. Gently swirl the plate every 15 minutes to prevent the media from aggregating at the edges of the plate and drying out. After an hour, add 10 units of DNase in one milliliter of 10X DNase buffer and add it to the wells.Incubate the plate at 37 degrees Celsius for 15 minutes to remove residual plasmid DNA. Now wash the plate once with one milliliter of growth medium, then add two milliliters of fresh media. Incubate the plate at 25 degrees Celsius for 24 to 36 hours.When incubation is complete, split the contents of a well into a new plate at a 1:2 ratio. Then collect the trophozoites from the remaining wells for further analysis. The Naegleria gruberi trophozoites were amoeboid and adherent to the culture flasks under standard culture conditions.Their incubation on ice resulted in round and non-adherent trophozoites. Incubation of the trophozoites in encystment media caused them to become encysted.

Closed Circular Extrachromosomal Ribosomal DNA (rDNA) Containing Element (CERE) Isolation from Transfected Naegleria Gruberi Trophozoites and PCR Analysis

To begin, add 200 microliters of 100 millimolar sodium chloride to a tube containing transfected trophozoites of naegleria gruberi. Centrifuge the tube at 600G for 10 minutes at 4 degrees Celsius. After removing the supernatant, resuspend the cell pellet in 100 microliters of 100 millimolar EDTA.Place the tube in a heat block for 15 minutes at 98 degrees Celsius to lyse the cells. Centrifuge the tube at 4 degrees Celsius for 10 minutes at top speed to pellet the debris. Transfer the supernatant into a new tube.Next, add 0.5 volumes of ammonium acetate, three volumes of absolute ethanol, and one microliter of glycogen to the supernatant. Invert the tube several times to mix, before incubating it at minus 80 degrees Celsius for 30 minutes or overnight. After incubation, centrifuge the tubes at room temperature for 10 minutes at 16, 000G.Remove the supernatant without disturbing the pellet, and add 200 microliters of 70%ethanol to wash it before centrifuging. Resuspend the dried pellet in sterile deionized water to achieve a trophozoite density of 10, 000 trophozoites per microliter. After PCR with primer specific for the transfected DNA and detection of the transformed trophozoites, add two microliters of 6X loading dye to the CERE samples.Then, load the dyed samples onto a 0.8%agarose gel. After electrofluorescing the gel for 1.5 to 2 hours, incubate it in DNA dye diluted in 100 milliliters of deionized water. Transfer the stained gel into deionized water for 20 minutes to destain it.Visualize the gel on an ultraviolet light system to document the results. PCR analysis showed that pGRUB was detected in transfected trophozoites through at least seven passages. pGEM was lost after the first passage, indicating that the empty plasmid was not retained by the amoebas.

334 Views.  Creighton University School of Medicine.  This protocol describes a methodology to transfect Naegleria gruberi trophozoites with a construct that is maintained throughout passaging trophozoites in vitro, as well as through encystment and excystment.

Using a CERE-Based Vector for Transfection of Naegleria gruberi Trophozoites

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