Name: forward genetic approaches in chlamydia trachomatis
Uploaded: 2013-10-23T18:30:00
Description: Watch this Scientific Journal Video about Forward Genetic Approaches in Chlamydia trachomatis at JoVE.com

1.. Kemisk mutagenese Bemærk: Vi fandt, at den replikative RB formen er mere modtagelig for kemisk mutagenese end EB formularen. Midt i cyklus (mellem 18.-20 HPI) er RB på det største antal forud for RB-EB overgang. Fordi Chlamydia trachomatis er en obligat intracellulær patogen, kan virkningerne af mutagen på værten sundhed begrænse bakteriel genopretning. Vero-celler blev fundet at være mere modstandsdygtige over for de negative virkninger af høje niveauer af EMS end andre ...

<ol>
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Denne metode opfylder de grundlæggende krav til genetisk analyse da det etablerer forbindelsen mellem genotyper og fænotyper. Vigtigere er, opnås dette uden hjælp af konventionelle molekylære værktøjer til rekombinant DNA-transformation og insertionel inaktivering af gener i bakterier, som ofte er et hastighedsbegrænsende trin i analysen af ​​genfunktion i ikke-model mikrober. Et kritisk trin er at sikre klonalitet af plak-oprensede mutanter. Krydskontaminering med vildtype eller...

De obligate intracellulære bakterien Chlamydia trachomatis regnskab for en anslået 2,8 millioner genitale infektioner om året i USA (Center for Disease Control) med tilhørende følgesygdomme såsom underlivsbetændelse, graviditet udenfor livmoderen, og infertilitet (1). Chlamydia-arter har en unik fysiologi med en bifasisk udviklingsmæssige cyklus bestående af to former: den infektiøse, men ikke-replikerende elementær krop (EB) og infektiøse men replikative reticulate legeme (RB). Infektion begynder med fastgørelsen af ​​EBS til epitelceller efterfulgt af endoctyotosis (2). Inden for en membran-bundet vacuole betegnet en optagelse, EBS differentiere i RB formular, som så replikater ved binær fission. Midt i cyklus, RBS overgange tilbage i EBS, som derefter bortvist i det ekstracellulære rum til at starte en ny omgang infektion, når værten celle lyserer (3). C. trachomatis errefraktær over for rutinemæssig manipulation med standard molekylærgenetiske redskaber, såsom målrettet genudskiftning, transposoner og transducerende fager, som har været centralt for de fleste studier i bakterielle genetik, Det er uklart, i hvilket omfang de enkelte Chlamydia gener bidrager til unddragelse af medfødte immunitet , næringsstof erhvervelse, udviklingsmæssige overgange, eller andre processer der er vigtige for patogenet overlevelse i en eukaryot vært (4). Følgelig dette patogen stadig dårligt karakteriseret trods sin klinisk betydning. Genomer af Chlamydia spp. er relativt lille (~ 1 Mb) (5) med flere arter og biovarer sekventeret bruge næste generation sekventering teknologier. Sammenlignende genom analyse WGS har givet enestående indblik i udviklingen af ​​chlamydia arter og deres tilpasning til mennesker (6-8), og til en vis grad har givet nogle oplysninger, som den potentielle funktion virulensfaktorer (9, 10). THan genetiske mangfoldighed vises af kliniske isolater giver ikke beslutningen forpligtet til systematisk at kortlægge funktionen af ​​de fleste virulensfaktorer, formentlig fordi mutationer i sådanne gener ville have været let vælges imod. Uden forstyrrende effekter fra naturlig udvælgelse, mutagen-induceret gen variation, kombineret med definerede analyser, der måler fejl i virulens, kan udvide det spektrum af mutationer, der kan adspurgte. Kemiske mutagener i særdeleshed, er nyttige, da de kan generere null, betinget hypomorphic (nedsat funktion) og hypermorphic (gevinst på funktion) alleler. Med ankomsten af ​​robuste næste generations genom-sekventering teknologier, kan sådanne mutationer let identificeres og kortlægges. På denne måde kan stærke associationer foretages mellem mutationer i et gen eller genetisk vej og en fælles fænotype, der muliggør anvendelse af forward genetiske metoder. Genom-sekvenser af kliniske stammer afslørede mosaicisme Between serovarer og loci for hyppig rekombination (11). Empiriske beviser for rekombination blev demonstreret gennem co-infektion af to forskellige antibiotikaresistente stammer og valg af dobbelt resistente rekombinant afkom, som blev åbenbaret for at have genetiske bidrag fra begge stammer (12, 13). Således genetisk udveksling mellem vildtype og mutantstammer i en co-infektion indstilling giver adskillelse af kemisk inducerede mutationer til at lokalisere genet påvirkede, der fører til den observerede fænotype. Her beskriver vi en metode til at udføre genetisk analyse i Chlamydia baseret på kemisk mutagenese, WGS, og et system til DNA udveksling i inficerede celler (14) (figur 1).

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	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)</td>
			<td>Life Technologies</td>
			<td>11995-073</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Cellgro</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl methanesulfonate (EMS)</td>
			<td>Sigma</td>
			<td>M0880</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclohexamide</td>
			<td>Sigma</td>
			<td>C4859-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Life Technologies</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Life Technologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) solution with 0.493 mM MgCl2 and 0.901 mM CaCl2 (PBS+MgCl2/CaCl2)</td>
			<td>Life Technologies</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M NaOH</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5x SPG buffer (1.25 M sucrose, 50 mM sodium phosphate, 25 mM glutamic acid)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SPG buffer (0.25 M sucrose, 10 mM sodium phosphate, 5 mM glutamic acid)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water (sterile, tissue culture grade)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2x DMEM (prepared from powder, buffered with 7.4 g/L Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>D7777</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonessential amino acids (NEAA)</td>
			<td>Life Technologies</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>1.2% GTG Agarose , autoclaved</td>
			<td>Lonzo</td>
			<td>50070</td>
			<td></td>
		</tr>
		<tr>
			<td>Genomic DNA purification kits</td>
			<td>Qiagen</td>
			<td>69504</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAzol</td>
			<td>Life Technologies</td>
			<td>10503-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (molecular biology grade)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>8 mM NaOH</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.1 M HEPES ( 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25 cm2 tissue culture flasks (T25 flasks)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>12-well tissue culture plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well tissue culture plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chemical safety hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biological safety hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#62;Centrifuge and adaptors for spinning tissue plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#62;Dissection microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorometer (Qubit)</td>
			<td>Invitrogen</td>
			<td>Q32866</td>
			<td></td>
		</tr>
		<tr>
			<td>Adaptive Focused Acoustics S220 instrument</td>
			<td>Covaris</td>
			<td></td>
			<td></td>
		</tr>
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forward genetic approaches in chlamydia trachomatis

Chlamydia trachomatis, den ætiologiske agens af seksuelt overførte sygdomme og øjeninfektioner, forbliver dårligt karakteriseret på grund af sin intractability til eksperimentel transformation med rekombinant DNA. Vi udviklede en metode til at udføre genetisk analyse i C. trachomatis trods manglen på molekylærgenetiske værktøjer. Vores fremgangsmåde involverer: i) kemisk mutagenese til hurtigt at generere omfattende biblioteker af genetisk definerede mutanter med forskellige fænotyper ii) hel-genom sekventering (WGS) at kortlægge de underliggende genetiske læsioner og finde sammenhænge mellem muterede gen (r) og.. en fælles fænotype,. iii) generation af rekombinante stammer gennem co-infektion af pattedyrceller med mutant og vildtype bakterier. Derfor var vi i stand til at etablere årsagssammenhænge genotyper og fænotyper. Koblingen af ​​kemisk-induceret gen variation og WGS at etablere korrelationsmaalinger genotype-fænotype foreninger Should være bredt anvendelig til den store liste over lægeuddannede og miljømæssigt vigtige mikroorganismer i øjeblikket uløselige til genetisk analyse.

Udsættelse for mutagen fører til indeslutninger, der vises uden bakterier, formentlig på grund af bakteriel celledød. Typisk vil serovar LGV-L2 helt lysere inficerede celler inden for 48 timer efter infektion, men behandling med mutagener kan udvide denne cyklus&gt; 90 timer. Omkring 10% af indeslutninger ventes at komme sig. I vores eksperimenter, behandlede inficerede Vero celler med 20 mg / ml EMS førte til et 99% fald i inddrivelse af infektiøse afkom i forhold til ubehandlede kontroller. Mutagen behandling f?...

Watch this Scientific Journal Video about Forward Genetic Approaches in Chlamydia trachomatis at JoVE.com

Forward Genetic Approaches in Chlamydia trachomatis

Forward Genetiske tilgange i<em&gt; Chlamydia trachomatis</em

Vi beskriver en metode til at udføre genetisk analyse i Chlamydia baseret på kemisk mutagenese og hele genomet sekventering. Desuden er et system til DNA-udveksling inden inficerede celler beskrevet, som kan anvendes til genetisk kortlægning. Denne metode kan være bredt anvendelig til mikrobielle systemer mangler transformation systemer og molekylærgenetiske værktøjer.

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its ...

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Immunology and Infection

Forward Genetic Approaches in Chlamydia trachomatis

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Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-ÃƒÅ½Ã‚Â³-Dependent Cell Autonomous Immunity

Forward Genetik skærme med makrofager til at identificere<em&gt; Toxoplasma gondii</em&gt; Gener Vigtigt for Modstand mod IFN-γ-Dependent Cell Autonome Immunitet

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

Udnytte den Antigen capsidet-Indbygning strategi for udvikling af adenovirusserotype 5-vektoriserede Vaccine Approaches

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Fag Phenomics: Fysiologiske tilgange til Karakterisere Novel virale proteiner

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

A Simple Red Blood Cell Lysis Method for the Establishment of B Lymphoblastoid Cell Lines

En simpel Red Blood Cell Lysis metode til fastsættelse af B lymfoblastoidcellelinier

A number of methods exist for the transformation of B lymphocytes by the Epstein Barr virus in vitro into immortalized cell lines. We have developed a new ...

An Efficient and Simple Method to Establish NK and T Cell Lines from Patients with Chronic Active Epstein-Barr Virus Infection

En effektiv og enkel metode til at etablere NK og T-cellelinier fra patienter med kronisk aktiv Epstein - Barr virusinfektion

A number of methods have been described to establish NK/T cell lines from patients with lymphoma or lymphoproliferative syndrome. These methods employed ...