Name: a genetic screen to isolate toxoplasma gondii host-cell egress mutants
Uploaded: 2012-02-08T13:00:00
Description: Watch this Scientific Journal Video about A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants at JoVE.com

Dette arbeidet ble finansiert av American Heart Association Scientist Development Grant 0635480N og National Institutes of Health forskningsstipend AI081220. BIC er støttet av en tempelridderne Eye Foundation forskningsstipend.

Oversikt Protokoller er forutsatt å først definere dosering av mutagen fører til en 70% drap av parasitter (protokoll 1). Den neste prosedyre for å berike de induserte utgående mutantene fra en mutagenized parasitt basseng (protokoll 2, figur 2). Dette etterfølges av en protokoll for å teste forekomst av utgående mutanter i anriket bassenget, eller å validere egress fenotypen i enkelte mutanter (protokoll 3). Endelig er en protokoll gis for å generere enkle parasitten kloner fra berik...

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<html> Den beskrives protokollen gir en effektiv metode for å isolere Toxoplasma mutanter med en egress defekt. Vi har lyktes i å isolere mutanter langs ulike stegene i egress vei, hvorav noen har en dobbel invasjon fenotype 13. Potensielle effekter på invasjon kan bestemmes ved hjelp av såkalt rød-grønn-analysen, som skiller invaderte fra ikke-invaderte parasitter ved differensial antistoff farging 23,24. For både invasjonen og egress analysene kan det være praktisk å uttrykke en ...

<table border="1"><tbody><tr><td> Navn på reagensen </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> ENU </td><td> Sigma-Aldrich </td><td> N3385 </td><td> 1 M Stock i DMSO, butikken ved -20 ° C </td></tr><tr><td> EMS </td><td> Sigma-Aldrich </td><td> M0880 </td><td> 1 M Stock i DMSO, butikken ved -20 ° C </td></tr><tr><td> Dekstransulfat </td><td> Sigma-Aldrich </td><td> D4911 </td><td></td></tr><tr><td> PDTC </td><td> Sigma-Aldrich </td><td> P8765 </td><td> 100 mm Stock i PBS </td></tr><tr><td> Diff Rask </td><td> EMD Kjemikalier </td><td> 65044-93 </td><td></td></tr><tr><td> Filterholderen </td><td> Cole-Parmer </td><td> 540100 </td><td></td></tr><tr><td> 3 mikrometer polykarbonat filter </td><td> WhatmanSchleicher &amp; Schuell </td><td> 110612 </td><td></td></tr><tr><td> Hemocytometer </td><td> Hausser Scientific </td><td> 1475 </td><td></td></tr><tr><td> CO 2 inkubatorer </td><td> Forskjellige produsenter </td><td></td><td> Fuktet, 5% CO 2, ved 35, 37 og 40 ° C </td></tr><tr><td> Fluorescens mikroskop </td><td> Forskjellige produsenter </td><td></td><td> Ideelt invertert, wide-felt med 63x eller 100x olje objektiv </td></tr></tbody></table> HBSSc (ifølge Svart m.fl. 11.): <ul><li> 98,0 ml Hanks Balanced Salt Solution (Hyclone katalognummer SH30588) </li><li> 100 mL 1M MgCl 2 (100 mm slutt) </li><li> 100 mL 1M CaCl 2 (100 mm slutt) </li><li> 2,0 ml 1M Hepes pH 7,3 (20 mm slutt) </li><li> 84 mg NaHCO 3 (10 mM slutt) </li></ul>

a genetic screen to isolate toxoplasma gondii host-cell egress mutants

Den utbredte, obligate intracellulære, protozo parasitten Toxoplasma gondii forårsaker opportunistisk sykdom i immuno-kompromitterte pasienter og forårsaker fødselsdefekter ved medfødt infeksjon. Den lytisk replikasjon syklusen er preget av tre stadier: 1. aktiv invasjon av en kjerneholdige vertscelle; to. replikering inne i vertscellen, 3. aktiv egress fra vertscellen. Mekanismen for egress blir i økende grad verdsatt som en unik, svært regulert prosess, som fortsatt er dårlig forstått på molekylært nivå. De signalveier underliggende egress har vært preget gjennom bruk av farmakologiske preparater som virker på ulike aspekter av banene 1-5. Som sådan har flere uavhengige utløsere av egress blitt identifisert som alle konvergerer på utgivelsen av intracellulær Ca 2 +, et signal som også er kritisk for vertscelle invasjon 6-8. Denne innsikten informert en kandidat gen tilnærming som førte til identifiseringen av anlegget som kalsium avhengig protein kinase (CDPK) involvert i egress 9. I tillegg har flere nylige gjennombrudd i forståelsen egress blitt gjort ved hjelp av (kjemisk) genetiske tilnærminger 10-12. Å kombinere vell av farmakologiske informasjon med økende genetisk tilgjengeligheten av Toxoplasma vi nylig etablert en skjerm tillater berikelse for parasitten mutanter med en defekt i vertscelle egress 13. Selv om kjemisk mutagenese bruke N-etyl-N-nitrosourea (ENU) eller etyl methanesulfonate (EMS) har vært brukt i flere tiår i studiet av Toxoplasma biologi 11,14,15, bare nylig har genetisk kartlegging av mutasjoner som ligger bak fenotyper bli rutine 16 -18. Videre, ved å generere temperaturfølsomme mutanter, kan viktige prosesser bli dissekert og de underliggende genene direkte identifisert. Disse mutantene oppfører seg som vill-type under ettergivende temperatur (35 ° C), men mislykkes i å proliferate ved den restriktive temperatur (40 ° C) som følge av mutasjon i spørsmålet. Her har vi illustrere en ny fenotypisk screening metode for å isolere mutanter med en temperatur-sensitiv egress fenotype 13. Utfordringen for utgående skjermer er å skille egressed fra ikke-egressed parasitter, som kompliseres av rask re-invasjon og generell Stickiness av parasittene til vertsceller. En tidligere etablert egress skjerm var basert på en tungvint serie biotinylation skritt for å skille intracellulær fra ekstracellulære parasitter 11. Denne metoden heller ikke generere betingede mutanter resulterer i svake fenotyper. Metoden beskrevet her overvinner den sterke tilknytning til egressing parasitter ved å inkludere en glykan konkurrent, dekstransulfat (DS), som hindrer parasitter fra stikker til vertscellen 19. Videre er ekstracellulære parasitter spesifikt drept av ved pyrrolidine dithiocarbamate (PDTC), som etterlater intracellulære parasitteruskadd 20. Derfor, med en ny fenotypisk skjermen for å spesifikt isolere parasitten mutanter med defekter i indusert egress, kan kraften av genetikk nå bli fullt utviklet for å avdekke molekylære mekanismene bak vertscellen egress.

Watch this Scientific Journal Video about A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants at JoVE.com

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Forward genetikk er en kraftig metode for å avdekke molekylære nivå av hvordan<em&gt; Toxoplasma</em&gt; Egresses fra sin vertscellen. Protokollene er gitt til kjemisk mutagenize parasitter, berike for mutanter med defekter i indusert egress, og validere fenotype av klonede mutanter.

En Screeningsteknikken å isolere Toxoplasma gondii Host-celle Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

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Immunology and Infection

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

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