Name: visualization of endoplasmic reticulum localized mrnas in mammalian cells
Uploaded: 2012-12-17T15:00:00
Description: Watch this Scientific Journal Video about Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells at JoVE.com

Dette arbejde blev støttet af tilskud fra National Science and Engineering Research Council of Canada til XAC og AFP

1. Forberedelse af Materialer til Extraction <ol><li> Fremstilling af celler <ol><li> Seed vævskulturceller på syrebehandlede dækglas i mindst en dag før forsøget. Vi bruger COS-7 eller U2OS, da disse to cellelinjer udviser robust produktion af udskilt protein og har et veldefineret ER. Bemærk, at for at opnå høj ekstraktionseffektivitet, må cellerne ikke overstige 80% konfluens på dækglasset på dagen for forsøget. </li><li> Hvis en bestemt eksogent mRNA bliver undersøgt, bliver cellerne transficeret én...

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Lokaliseringen af ​​mRNA&#39;er til forskellige subcellulære steder, gennem interaktion af udskrifter med mRNA lokalisering proteiner, er et udbredt fænomen vigtig for en korrekt sortering af proteiner til deres endelige bestemmelsessted, og for finjustering af genekspression til de lokale krav til et subcellulært region 19,20. Anvendelse af assayet beskrevet her, vi revisited hvorvidt mRNA&#39;er, der koder sekretoriske proteiner kan opretholdes på ER i nærvær eller fra...

I eukaryoter secerneret mRNA kodende og membranproteiner kan målrettes til ER co-translationelt ved signalgenkendelse partikel 1,2 og kan opretholdes på ER via direkte interaktioner mellem ribosomer og translocons under oversættelsen 3,4. Men hvorvidt mRNA&#39;er kan målrettes og vedligeholdes på skadestuen uafhængig af enten ribosomer eller oversættelser var uklar indtil for ganske nylig. Tidligere undersøgelser har forsøgt at løse, om der er translation-uafhængig mRNA samarbejde med ER via et trådløst fraktioneringsteknikker. Fordi barske kemiske betingelser skulle adskille ribosomer fra ER afledte vesikler, som vil kunne skabe potentielt følsomme mRNA-ER forening, disse undersøgelser var inkonklusive, der beviser for 5-8 og imod 9,10 ribosomal-uafhængig forankring af mRNA til ER . At omgå disse problemer har vi udviklet en protocol at isolere og billedet ER-bundne mRNA&#39;er. Denne procedure indebærer en mild ekstraktionsbehandling, der effektivt fjerner al den cytoplasmiske indhold af cellen (herunder ikke ER-bundne mRNA&#39;er) samtidig bevare ER-morfologi og alle dertil hørende molekyler. Under anvendelse af denne protokol, har vi vist, at en delmængde af mRNA&#39;er målrettes og derefter holdt på ER uafhængigt af ribosomer eller oversættelse 11.

visualization of endoplasmic reticulum localized mrnas in mammalian cells

I eukaryoter. Fleste af messenger RNA&#39;er (mRNA&#39;er), der koder for secernerede og membran-proteiner er lokaliseret til overfladen af ​​det endoplasmatiske reticulum (ER) Dog kan visualiseringen af ​​disse mRNA&#39;er være udfordrende. Dette er især tilfældet, når kun en brøkdel af mRNA&#39;en er ER-associeret og deres fordeling til dette organel hindres af ikke-målet (dvs. &quot;fri&quot;) transkripter. For at overvåge ER-associerede mRNA&#39;er, har vi udviklet en fremgangsmåde, hvor celler behandles med en kort eksponering for en digitonin ekstraktionsmiddel, der selektivt permeabilizes plasmamembranen, og således fjerner de cytoplasmatiske indhold, at man samtidig opretholder integriteten af ​​ER . Når denne metode kombineres med fluorescerende in situ hybridisering (FISH), kan man tydeligt visualisere ER-bundne mRNA&#39;er ved hjælp af fluorescensmikroskopi. Brug af denne protokol graden af ​​ER-forening for enten bulk-poly (A) udskrifter eller specifikke mRNA&#39;er kan vurderesog endog kvantificeres. Ved fremgangsmåden kan man anvende dette assay til at undersøge karakteren af ​​mRNA-ER-interaktioner.

At bestemme procentdelen af mRNA, der er ER-associeret, COS-7-celler, der var transficeret med plasmider, der indeholdt placental alkalisk phosphatase (ALPP) eller cytochrom P450-8B1 (CYP8B1) blev enten ekstraheret med digitonin og derefter fikseret, eller direkte fastgjort (se figur 1, sammenlign &quot;Unextracted Ctrl&quot; til &quot;Ekstraheret Ctrl&quot;). Den ikke-nuklear fluorescens blev kvantificeret i begge prøver, og fraktionen af ER-associerede mRNA blev beregnet (fi...

Watch this Scientific Journal Video about Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells at JoVE.com

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

Her beskriver vi en metode til at synliggøre endoplasmatiske reticulum-associerede mRNA&#39;er i mammale vævskulturceller. Denne teknik involverer selektiv permeabilisering af plasmamembranen med digitonin til fjernelse af cytoplasmatisk indhold efterfulgt af fluorescerende<em&gt; In situ</em&gt; Hybridisering til påvisning af enten bulk-poly (A) mRNA eller specifikke transkripter.

Visualisering af endoplasmatiske reticulum lokaliserede mRNA&#39;er i pattedyrceller

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

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