Dessa studier finansierades delvis av ett anslag från George F. Haddix Fund vid Creighton University (KMD). Figur 1 genereras i biorender.com och figur 3 genereras i benchling.com.

Detaljerna om arten, reagenserna och utrustningen som används i denna studie listas i materialtabellen. Sekvensen för pGRUB-konstruktionen med 17 004 baspar finns i tilläggsfil 1.
1. Odling av trofozoiter
<ol><li>Tina frysta N. gruberi trophozoites vid 37 °C i 3 min.</li>
	<li>Inokulera trofozoiter i T25-kolvar i modifierat pepton-/jästextrakt/nukleinsyra/folsyra/hemin med 10 % fetal kalvserummedia (PYNFH) plus 2 % buffert (din...

Användning av en CERE-baserad vektor för transfektion av Naegleria gruberi trophozoites

<ol>
	<li>De Jonckheere, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+do+we+know+by+now+about+the+genus+Naegleria.">What do we know by now about the genus Naegleria.</a> Exp Parasitol. 145 (Suppl), S2-S9 (2014).</li><li>De Jonckheere, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+century+of+research+on+the+Amoeboflagellate+genus+Naegleria.">A century of research on the Amoeboflagellate genus Naegleria.</a> Acta Protozool. 41, 309-342 (2002).</li><li>Fulton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+differentiation+in+Naegleria+gruberi.">Cell differentiation in Naegleria gruberi.</a> Annu Rev Microbiol. 31, 597-629 (1977).</li><li>Grace, E., Asbill, S., Virga, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naegleria+fowleri:+Pathogenesis,+diagnosis,+and+treatment+options.">Naegleria fowleri: Pathogenesis, diagnosis, and treatment options.</a> AAC. 59 (11), 6677-6681 (2015).</li><li>Moseman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Battling+brain-eating+amoeba:+enigmas+surrounding+immunity+to+Naegleria+fowleri.">Battling brain-eating amoeba: enigmas surrounding immunity to Naegleria fowleri.</a> PLOS Pathog. 16 (4), e1008406 (2020).</li><li>Marciano-Cabral, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+Naegleria+spp.">Biology of Naegleria spp.</a> Microbiol Rev. 52 (1), 114-133 (1988).</li><li>Pi&#241;ero, J. E., Oma&#241;a-Molina, M., Ch&#225;vez-Mungu&#237;a, B., Lorenzo-Morales, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naegleria+fowleri.">Naegleria fowleri.</a> Trends Parasitol. 35 (10), 848-849 (2019).</li><li>Zysset-Burri, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+identification+of+pathogenicity+factors+of+the+free-living+amoeba+Naegleria+fowleri.">Genome-wide identification of pathogenicity factors of the free-living amoeba Naegleria fowleri.</a> BMC Genomics. 15, 496-510 (2014).</li><li>Liechti, N., Sch&#252;rch, N., Bruggmann, R., Wittwer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+of+Naegleria+lovaniensis,+the+basis+for+a+comparative+approach+to+unravel+pathogenicity+factors+of+the+human+pathogenic+amoeba+N.+fowleri.">The genome of Naegleria lovaniensis, the basis for a comparative approach to unravel pathogenicity factors of the human pathogenic amoeba N. fowleri.</a> BMC Genom. 19, 654-665 (2018).</li><li>Liechti, N., Sch&#252;rch, N., Bruggmann, R., Wittwer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanopore+sequencing+improves+the+draft+genome+of+the+human+pathogenic+amoeba+Naegleria+fowleri.">Nanopore sequencing improves the draft genome of the human pathogenic amoeba Naegleria fowleri.</a> Sci Reports. 9, 16040-16049 (2019).</li><li>Fritz-Laylin, L. K., Ginger, M. L., Walsh, C., Dawson, S. C., Fulton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Naegleria+genome:+A+free-living+microbial+eukaryote+lends+unique+insights+into+core+eukaryotic+cell+biology.">The Naegleria genome: A free-living microbial eukaryote lends unique insights into core eukaryotic cell biology.</a> Res Microbiol. 162, 607-618 (2011).</li><li>Clark, C. G., Cross, G. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=rRNA+genes+of+Naegleria+gruberi+are+carried+exclusively+on+a+14-kilobase-pair+plasmid.">rRNA genes of Naegleria gruberi are carried exclusively on a 14-kilobase-pair plasmid.</a> Mol Cell Biol. 7 (9), 3027-3031 (1987).</li><li>Clark, C. G., Cross, G. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+ribosomal+RNA+genes+are+a+general+feature+of+schizopyrenid+amoebae.">Circular ribosomal RNA genes are a general feature of schizopyrenid amoebae.</a> J Protozool. 35 (2), 326-329 (1988).</li><li>Dao, F. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identify+origin+of+replication+in+Saccharomyces+cerevisiae+using+two-step+feature+selection+technique.">Identify origin of replication in Saccharomyces cerevisiae using two-step feature selection technique.</a> Bioinformatics. 35 (12), 2075-2083 (2019).</li><li>Mullican, J. C., Chapman, N. M., Tracy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+genome+sequence+of+the+circular+extrachromosomal+element+of+Naegleria+gruberi+strain+EGB+ribosomal+DNA.">Complete genome sequence of the circular extrachromosomal element of Naegleria gruberi strain EGB ribosomal DNA.</a> Genome Announc. 6 (6), e00020-e00021 (2018).</li><li>Mullican, J. C., Drescher, K. M., Chapman, N. M., Tracy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+genome+sequence+of+the+Naegleria+fowleri+(strain+LEE)+closed+circular+extrachromosomal+ribosomal+DNA+element.">Complete genome sequence of the Naegleria fowleri (strain LEE) closed circular extrachromosomal ribosomal DNA element.</a> Microbiol Resource Announce. 9 (49), e01055-e01020 (2020).</li><li>Nguyen, B. T., Chapman, N. M., Tracy, S., Drescher, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+extrachromosomal+elements+of+the+Naegleria+amoebae:+How+little+we+know.">The extrachromosomal elements of the Naegleria amoebae: How little we know.</a> Plasmid. 115, 102567 (2021).</li><li>Nguyen, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+sequence+of+the+closed+circular+extrachromosomal+element+(CERE)+of+Naegleria+australiensis+De+Jonckheere+(strain+PP+397).">Complete sequence of the closed circular extrachromosomal element (CERE) of Naegleria australiensis De Jonckheere (strain PP 397).</a> Microbiol Resource Announce. 12, e0032123 (2023).</li><li>Nguyen, B. T., Chapman, N. M., Stessman, H. A. F., Tracy, S., Drescher, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+sequence+of+the+closed+circular+extrachromosomal+element+of+Naegleria+jadini+Willaert+and+Ray+(Strain+ITMAP400).">Complete sequence of the closed circular extrachromosomal element of Naegleria jadini Willaert and Ray (Strain ITMAP400).</a> Microbiol Resource Announce. 12 (4), e0006123 (2023).</li><li>Nguyen, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+sequence+of+the+closed+circular+extrachromosomal+element+(CERE)+of+Naegleria+pringsheimi+De+Jonckheere+(strain+Singh).">Complete sequence of the closed circular extrachromosomal element (CERE) of Naegleria pringsheimi De Jonckheere (strain Singh).</a> Microbiol Resource Announce. 13 (4), (2024).</li><li>Mullican, J. C., Chapman, N. M., Tracy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+single+origin+of+replication+in+the+Naegleria+gruberi+extrachromosomal+DNA+element.">Mapping the single origin of replication in the Naegleria gruberi extrachromosomal DNA element.</a> Protist. 170, 141-152 (2019).</li><li>Sohn, H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+liquid+media+for+encystation+of+pathogenic+free-living+amoebae.">Efficient liquid media for encystation of pathogenic free-living amoebae.</a> Korean J Parasitol. 55 (3), 233-238 (2017).</li><li>Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypan+blue+exclusion+test+of+cell+viability.">Trypan blue exclusion test of cell viability.</a> Curr Protoc Immunol. 111, A3.B.1-A3.B.3 (2001).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+Chain+Reaction:+Basic+protocol+plus+troubleshooting+and+optimization+strategies.">Polymerase Chain Reaction: Basic protocol plus troubleshooting and optimization strategies.</a> J Vis Exp. 63, e3998 (2012).</li><li>Jeong, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+nfa1+gene+cloned+from+pathogenic+Naegleria+fowleri+in+non-pathogenic+N.+gruberi+enhances+cytotoxicity+against+CHO+target+cells+in+vitro.">Expression of the nfa1 gene cloned from pathogenic Naegleria fowleri in non-pathogenic N. gruberi enhances cytotoxicity against CHO target cells in vitro.</a> Infect Immun. 73 (7), 4098-4105 (2005).</li><li>Clark, C. G., Cross, G. A. M., De Jonckheere, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+evolutionary+divergence+in+the+genus+Naegleria+by+analysis+of+ribosomal+DNA+plasmid+restriction+patterns.">Evaluation of evolutionary divergence in the genus Naegleria by analysis of ribosomal DNA plasmid restriction patterns.</a> Mol Biochem Parasitol. 34 (3), 281-296 (1989).</li><li>De Jonckheere, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+variation+in+the+ribosomal+internal+transcribed+spacers,+including+the+5.8S+rDNA,+of+Naegleria+spp.">Sequence variation in the ribosomal internal transcribed spacers, including the 5.8S rDNA, of Naegleria spp.</a> Protist. 149 (3), 221-228 (1998).</li><li>Rawal, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+prediction+of+G4+DNA+as+regulatory+motifs:+Role+in+Escherichia+coli+global+regulation.">Genome-wide prediction of G4 DNA as regulatory motifs: Role in Escherichia coli global regulation.</a> Genome Res. 16 (5), 644-655 (2006).</li><li>Yadav, V. K., Abraham, J. K., Mani, P., Kulshrestha, R., Chowdhury, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=QuadBase:+Genome-wide+database+of+G4+DNA+-+occurrence+and+conservation+in+human,+chimpanzee,+mouse+and+rat+promoters+and+146+microbes.">QuadBase: Genome-wide database of G4 DNA - occurrence and conservation in human, chimpanzee, mouse and rat promoters and 146 microbes.</a> Nucleic Acids Res. 36, D381-D385 (2007).</li></ol>

Inga finansiella intressekonflikter har deklarerats.

Protokollet som beskrivs här är mycket enkelt, även om varje konstruktion sannolikt kommer att kräva en viss grad av optimering, särskilt av förhållandet mellan DNA-transfektionsreagens, beroende på konstruktionens karaktär och vilken art av Naegleria som används. Vi har bara testat ett kommersiellt tillgängligt transfektionsreagens med detta protokoll, men det är troligt att flera andra kan vara effektiva. Med tanke på att en fullängdsklon av CERE används, som innehåller ett funktionellt ursprun...

Släktet Naegleria innehåller över 45 arter, även om det är osannolikt att alla medlemmar av artenhar identifierats. Naegleria kan existera i olika former: som trofozoiter (amöbor), som flagellater eller, när resurserna är starkt begränsade, som cystor 1,2,3,4. Naegleria-släktet är känt för sin särskilt farliga art, Naegleria fowleri, känd som "den hjärnätande amöban" (granskad i 1,2,3,4,5,6,7), som är orsaken till den nästan universellt dödliga primära amöba-meningoencefaliten.
Naegleria kodar sitt rDNA på CERE som ligger i nukleolen. Fullständig sekvensering av tre Naegleria-arters genom bekräftar frånvaron av rDNA i det nukleära genomet 8,9,10,11. Baserat på de begränsade CERE-sekvenserna i full längd varierar CERE från cirka 10 kbp till 18 kbp i längd. CERE för varje art av Naegleria har en enda rDNA-cistron (som innehåller ribosomalt DNA 5,8, 18 och 28S) på var och en av cirka 4 000 CERE per cell 12,13,14. Förutom rDNA har varje CERE en stor icke-rDNA-sekvens (NRS). CERE-storlekarna varierar mellan olika arter; Skillnaderna beror främst på den varierande längden på NRS, eftersom rDNA-sekvenserna är mycket bevarade över hela släktet 1,15,16,17,18,19,20. Kartläggningen av ett enda ursprung för DNA-replikation inom N. gruberi CERE NRS21 ger starkt stöd för hypotesen att Naegleria CERE replikerar oberoende av det nukleära genomet.
N. gruberi är en icke-patogen amöba som ofta används för att studera Naegleria-biologi . Vi utvecklade en metod för att transformera N. gruberi trophozoites med en CERE-klon från samma art för att testa hypotesen att Naegleria amoebae tolererar och självständigt replikerar CERE inom trofozoiterna. Detta åstadkoms genom att omvandla N. gruberi med en fullängdsklon av N. gruberi CERE till trofozoiter och följa ödet för donatorns CERE-klon genom polymeraskedjereaktion (PCR). En allmän översikt över protokollet visas i figur 1. De data som presenteras här visar att den donerade klonen kan detekteras genom minst sju passager i vävnadskulturen, samt bibehållas genom incystment och excystment. Dessa studier ligger till grund för ett sätt att dissekera hur CERE replikerar, samt utforska dess användning som en vektor för att transfektera 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

Alla ribosomala gener hos Naegleria trophozoites bevaras i ett slutet cirkulärt extrakromosomalt ribosomalt DNA (rDNA) som innehåller element (CERE). Även om lite är känt om CERE, visar en fullständig genomsekvensanalys av tre Naegleria-arter tydligt att det inte finns några rDNA-cistroner i det nukleära genomet. Dessutom har ett enda DNA-ursprung för replikation kartlagts i N. gruberi CERE, vilket stöder hypotesen att CERE replikerar oberoende av det nukleära genomet. Denna CERE-egenskap tyder på att det kan vara möjligt att använda CERE för att introducera främmande proteiner i Naegleria trophozoiter. Som ett första steg i att utforska användningen av en CERE som en vektor i Naegleria utvecklade vi ett protokoll för att transfektera N. gruberi med en molekylär klon av N. gruberi CERE klonad till pGEM7zf+ (pGRUB). Efter transfektion påvisades pGRUB lätt hos N. gruberi trophozoites under minst sju passager, samt genom incystering och excystering. Som en kontroll transfekterades trofozoiter med ryggradsvektorn, pGEM7zf+, utan N. gruberi-sekvenserna (pGEM). pGEM detekterades inte efter den första passagen efter transfektion till N. gruberi, vilket tyder på dess oförmåga att replikera i en eukaryot organism. Dessa studier beskriver ett transfektionsprotokoll för Naegleria trophozoites och visar att den bakteriella plasmidsekvensen i pGRUB inte hämmar framgångsrik transfektion och replikation av den transfekterade CERE-klonen. Dessutom kommer detta transfektionsprotokoll att vara avgörande för att förstå den minimala sekvensen av CERE som driver dess replikation i trofozoiter, samt för att identifiera regulatoriska regioner i den icke-ribosomala sekvensen (NRS).

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.

Författare i fokus: Att förstå utbredningen av slutet cirkulärt extrakromosomalt rDNA innehållande element (CERE) i Naegleria gruberi

Transfektion av en molekylär klon av Naegleria gruberi rDNA till 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 av trofozoiter som har transfekterats med pGRUB visar att den transfekterade CERE detekteras genom minst sju passager av trofozoiterna, samt incystment och excystment (Figur 4). De primrar som används i PCR-glödgningen till både pGEM-vektorn och CERE-sekvensen, vilket säkerställer att PCR inte detekterar nativ CERE. PCR efter transfektion av pGEM till trofozoiter indikerade att pGEM var negativt (Figur 4), vilket indikerar att den tomma plasmiden inte h...

Detta protokoll beskriver en metod för att transfektera Naegleria gruberi trophozoiter med en konstruktion som bibehålls genom passerande trofozoiter in vitro, såväl som genom incystment och excystment.

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

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Research

JoVE Journal

Biology

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

316 Views.  Creighton University School of Medicine. 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...