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 Naegleria gruberi 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><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>&lt;em&gt;N. gruberi&lt;/em&gt;</td><td>ATCC</td><td>30224</td><td></td></tr><tr><td>Nucleic Acid</td><td>Chem Impex Int&amp;rsquo;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).

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 ...

Author Spotlight: Understanding the Propagation of Closed Circular Extrachromosomal rDNA Containing Element (CERE) in Naegleria gruberi

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.

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 ...

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

Research

JoVE Journal

Biology

558 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.

Video: Author Spotlight: Understanding the Propagation of Closed Circular Extrachromosomal rDNA Containing Element (CERE) in Naegleria gruberi

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFR&#945;, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80&deg;C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.

This technique demonstrates an efficient way to prepare replication-defective retroviral stocks encoding a human oncogene, and subsequently used for induction of myeloproliferative disease in the mouse model.

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, ...

Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in vivo are considered to be of the monocyte lineage2. Certain strains of MVV can replicate in other cell types, however3,4. The green fluorescent protein is a commonly used marker for studying lentiviruses in living cells. We have inserted the egfp gene into the gene for dUTPase of MVV. The dUTPase gene is well conserved in most lentivirus strains of sheep and goats and has been shown to be important in replication of CAEV5. However, dUTPase has been shown to be dispensable for replication of the molecular clone of MVV used in this study both in vitro and in vivo6. MVV replication is strictly confined to cells of sheep or goat origin. We use a primary cell line from the choroid plexus of sheep (SCP cells) for transfection and propagation of the virus7. The fluorescent MVV is fully infectious and EGFP expression is stable over at least 6 passages8. There is good correlation between measurements of TCID50 and EGFP. This virus should therefore be useful for rapid detection of infected cells in studies of cell tropism and pathogenicity in vitro and in vivo8.

We describe a molecular clone of maedi-visna virus that expresses GFP and is fully infectious. Replication of this virus can be detected by using fluorescence microscopy and flow cytometry.

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in ...

Immunology and Infection

Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. Recent findings revealed that TERRA molecules also interact with internal chromosomal regions to regulate gene expression in mouse embryonic stem (ES) cells. In line with this evidence, RNA fluorescence in situ hybridization (RNA-FISH) analyses have shown that only a subset of TERRA transcripts localize at chromosome ends. A better understanding of the dynamics of TERRA molecules will help define their function and mechanisms of action. Here, we describe a method to label and visualize single-telomere TERRA transcripts in cancer cells using the MS2-GFP system. To this aim, we present a protocol to generate stable clones, using the AGS human stomach cancer cell line, containing MS2 sequences integrated at a single subtelomere. Transcription of TERRA from the MS2-tagged telomere results in the expression of MS2-tagged TERRA molecules that are visualized by live-cell fluorescence microscopy upon co-expression of a MS2 RNA-binding protein fused to GFP (MS2-GFP). This approach enables researchers to study the dynamics of single-telomere TERRA molecules in cancer cells, and it can be applied to other cell lines.

Here, we present a protocol to generate cancer cell clones containing a MS2 sequence tag at a single subtelomere. This approach, relying on the MS2-GFP system, enables visualization of the endogenous transcripts of telomeric repeat-containing RNA (TERRA) expressed from a single telomere in living cells.

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in ...

Genetics

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the causative agent of Legionnaires' pneumonia, gains a pathogenic advantage over in vitro cultured bacteria when first harvested from protozoan cells prior to infection of mammalian macrophages. This suggests that important virulence factors may not be properly expressed in vitro. We have developed a tractable system for priming L. pneumophila through its natural protozoan host Acanthamoeba castellanii prior to mammalian cell infection. The contribution of any virulence factor can be examined by comparing intracellular growth of a mutant strain to wild-type bacteria after protozoan priming. GFP-expressing wild-type and mutant L. pneumophila strains are used to infect protozoan monolayers in a priming step and allowed to reach late stages of intracellular growth. Fluorescent bacteria are then harvested from these infected cells and normalized by spectrophotometry to generate comparable numbers of bacteria for a subsequent infection into mammalian macrophages. For quantification, live bacteria are monitored after infection using fluorescence microscopy, flow cytometry, and by colony plating. This technique highlights and relies on the contribution of host cell-dependent gene expression by mimicking the environment that would be encountered in a natural acquisition route. This approach can be modified to accommodate any bacterium that uses an intermediary host as a means for gaining a pathogenic advantage.

This technique provides a method to harvest, normalize and quantify intracellular growth of bacterial pathogens that are pre-cultivated in natural protozoan host cells prior to infections of mammalian cells. This method can be modified to accommodate a wide variety of host cells for the priming stage as well as target cell types.

Many intracellular bacterial pathogens use freshwater protozoans as a natural reservoir for proliferation in the environment. Legionella pneumophila, the ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Transient Transduction of the Strobilated Forms of Echinococcus granulosus

Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm harbored in the intestine of canines. There is an urgent need for applied genetic research to understand the mechanisms of pathogenesis and disease control and prevention. However, the lack of an effective gene evaluation system impedes direct interpretation of the functional genetics of cestode parasites, including the Echinococcus species. The present study demonstrates the potential of lentiviral gene transient transduction in the metacestode and strobilated forms of E. granulosus. Protoscoleces (PSCs) were isolated from hydatid cysts and transferred to specific biphasic culture media to develop into strobilated worms. The worms were transfected with harvested third-generation lentivirus, along with HEK293T cells as a transduction process control. A pronounced fluorescence was detected in the strobilated worms over 24 h and 48 h, indicating transient lentiviral transduction in E. granulosus. This work presents the first attempt at lentivirus-based transient transduction in tapeworms and demonstrates the promising outcomes with potential implications in experimental studies on flatworm biology.

Transient Transduction of the Strobilated Forms of Echinococcus granulosus

We describe a rapid transient transduction technique in different developmental stages of Echinococcus granulosus using third-generation lentiviral vectors.

Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm ...

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted area of the brain, neural stem cells proliferate and constantly generate neuroblasts that migrate tangentially in chains along the rostral migratory stream (RMS) to reach the olfactory bulb (OB). Once in the OB, neuroblasts switch to radial migration and then differentiate into mature neurons able to incorporate into the preexisting neuronal network. Proper neuroblast migration is a fundamental step in neurogenesis, ensuring the correct functional maturation of newborn neurons. Given the ability of SVZ-derived neuroblasts to target injured areas in the brain, investigating the intracellular mechanisms underlying their motility will not only enhance the understanding of neurogenesis but may also promote the development of neuroregenerative strategies.
This manuscript describes a detailed protocol for the transfection of primary rodent RMS postnatal neuroblasts and the analysis of their motility using a 3D in vitro migration assay recapitulating their mode of migration observed in vivo. Both rat and mouse neuroblasts can be quickly and efficiently transfected via nucleofection with either plasmid DNA, small hairpin (sh)RNA or short interfering (si)RNA oligos targeting genes of interest. To analyze migration, nucleofected cells are reaggregated in &#39;hanging drops&#39; and subsequently embedded in a three-dimensional matrix. Nucleofection per se does not significantly impair the migration of neuroblasts. Pharmacological treatment of nucleofected and reaggregated neuroblasts can also be performed to study the role of signaling pathways involved in neuroblast migration.

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Neuroblast migration is a crucial step in postnatal neurogenesis. The protocol described here can be used to investigate the role of candidate regulators of neuroblast migration by employing DNA/small hairpin RNA (shRNA) nucleofection and a 3D migration assay with neuroblasts isolated from the rodent postnatal rostral migratory stream.

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

Neuroscience

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of Plasmodium deposited by mosquitoes in the skin of vertebrate hosts undergoes a phase of mandatory development in the liver before initiating clinical malaria. We know little about the biology of Plasmodium development in the liver; access to the sporozoite stage and the ability to genetically modify such sporozoites are critical tools for studying the nature of Plasmodium infection and the resulting immune response in the liver. Here, we present a comprehensive protocol for the generation of transgenic Plasmodium berghei sporozoites. We genetically modify blood-stage P. berghei and use this form to infect Anopheles mosquitoes when they take a blood meal. After the transgenic parasites undergo development in the mosquitoes, we isolate the sporozoite stage of the parasite from the mosquito salivary glands for in vivo and in vitro experimentation. We demonstrate the validity of the protocol by generating sporozoites of a novel strain of P. berghei expressing the green fluorescent protein (GFP) subunit 11 (GFP11), and show how it could be used to investigate the biology of liver-stage malaria.

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is transmitted through inoculation of the sporozoite stage of Plasmodium by infected mosquitoes. Transgenic Plasmodium has allowed us to understand the biology of malaria better and has contributed directly to malaria vaccine development efforts. Here, we describe a streamlined methodology to generate transgenic Plasmodium berghei sporozoites.

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of ...

Retroviral Transduction of Guide RNA for Gene Editing in Th17 Differentiation

Retroviral CRISPR/Cas9-Mediated Gene Targeting for the Study of Th17 Differentiation in Vitro

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells can be stimulated with antigens and well-defined cytokine cocktails in vitro to mimic Th17 cell differentiation in vivo. Research has been conducted extensively on the Th17 differentiation regulation mechanisms using the in vitro Th17 polarization assay.
Conventional Th17 polarization methods typically involve obtaining na&#239;ve CD4 T cells from genetically modified mice to study the effects of specific genes on Th17 differentiation and function. These methods can be time-consuming and costly and may be influenced by cell-extrinsic factors from the knockout animals. Thus, a protocol using retroviral transduction of guide RNA to introduce gene knockout in CRISPR/Cas9 knockin primary mouse T cells serves as a very useful alternative approach. This paper presents a protocol to differentiate na&#239;ve primary T cells into Th17 cells following retroviral-mediated gene targeting, as well as the subsequent flow cytometry analysis methods for assaying infection and differentiation efficiency.

We present a protocol for retroviral transduction of guide RNA into primary T cells from Cas9 transgenic mice, providing an efficient alternative for gene editing in studying Th17 differentiation.

T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells ...

Nucleofection of Parasite Forms: An Electroporation-Based Transfection Method to Deliver Fluorescent Protein Encoding-Plasmids Into Sporozoite Nucleus

Source: Duan, C., et al. <a href="https://www.jove.com/v/60552">Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites.</a> J. Vis. Exp. (2020)
In this video, we describe nucleofection, an electroporation-based transfection procedure to deliver fluorescent reporter protein-encoding plasmid DNA into sporozoite form of the chicken Eimeria parasite.

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

Summary

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Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

Tractable Mammalian Cell Infections with Protozoan-primed Bacteria

Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Transient Transduction of the Strobilated Forms of Echinococcus granulosus

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Generating Genetically Modified Plasmodium berghei Sporozoites

Retroviral CRISPR/Cas9-Mediated Gene Targeting for the Study of Th17 Differentiation in Vitro

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