Name: stereotactic injection of microrna-expressing lentiviruses to the mouse hippocampus ca1 region and assessment of the behavioral outcome
Uploaded: 2013-06-10T12:00:00
Description: Watch this Scientific Journal Video about Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome Video at JoVE.com

Denne undersøgelse er blevet støttet af Edmond og Lily Safra Center for Brain Sciences (SB stipendium), The Legacy Heritage Biomedical Science Partnership Program for Israel Science Foundation (Grant No 378/11, til HS) og den tyske israelske Foundation for Scientific Forskning og Udvikling (GIF) (Grant No 1093-32.2/2010 til HS).

1.. Lentivirus Forberedelse <ol><li> Grow HEK 293FT celler til 90% sammenflydning. </li><li> På dagen for transfektion ændring cellemedium til serumfrit DMEM suppleret med 1 mM glutamin og 50 mg / ml penicillin-streptomycin. </li><li> Co-transficere cellerne med en pLKO.1-Puro vektor og med plasmider der koder for delta R8.2 og VSV-G-dele og miRNA af interesse, ved anvendelse af 10 pi 1 mg / ml polyethylenimin som en bærer 9.. </li><li> Saml emballerede lentivira ved 24 timer og 48 timer efter transfekti...

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Stereotaktisk injektion af en lentivirus er en relativt hurtig metode til in vivo vurdering af både op-eller ned-regulering af de forskellige gener og miRNA. Det vigtigste alternativ er en gensplejset mus, der er en meget mere omstændelig og tidskrævende teknik, så direkte lentivirus indsprøjtning. Desuden, den opregulering i lentivirus injektion, sker på et bestemt tidspunkt i den voksne mus og omfatter ikke nogen mulighed for utætheder under udvikling, da det oftest er tilfældet i gensplejsede mus, de...

Rollen af ​​bestemte miRNA i nervesystemet fungerer er for nylig blevet udfordret af lentiviral injektion i flere studier. MiRNA har vist sig at være afgørende for at fastholde og re-forme synapse struktur 1, synaptogenesis 2 og synapse remodellering og vedligeholdelse 3.. Disse undersøgelser tyder på, at miRNA er involveret via multileveled regulerende virkninger i både udformningen op og i at opretholde den vigtigste resultat af nervesystemet, kognitive funktion. Stereotaktisk injektion af lentivirus partikler i specifikke områder i gnaverhjerne muliggør søgning efter ændringer i synapse morfologi og neuronal aktivitet, og blev brugt til oprettelse af funktionelle betydning overudtrykt udskrifter 4,5. Direkte smitte af neuroner på veldefinerede områder i hjernen med miRNA-udtrykkende lentivira kan være impliceret i undersøgelser af aldring, hjernesygdomme og neurodegeneration, Undersøgelser af miRNAs i realm af adfærdsregulering 6-8 er på et langt mindre fremskreden tilstand og stereotaktisk injektion af miRNA-udtrykkende lentivirusinfektioner partikler efterfulgt af adfærdsmæssige test kan være nyttige til sådanne formål. Et væsentligt mere arbejdskrævende metode til at inducere over-eller under-ekspression involverer gensplejsede knock-eller knockout mus. Genetiske systemer kan yderligere tillade betinget og tidsmæssig kontrol på ekspression (f.eks Cre-Lox, Tet systemer), men disse næppe tilbyde den rumlige specificitet injektionsproceduren, og der er næsten altid en vis mængde af utætheder. Desuden har de tekniske procedurer ikke kræver operation, og kan bruges på tværs laboratorier med relativ god reproducerbarhed, men de er langsommere og kræver langt mere arbejdskraft og økonomiske ressourcer. Desuden har den tidsmæssige styring af over-eller under-ekspression i injicerede mus er langt mere præcist i forhold til genetisk modificerede mus.

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stereotactic injection of microrna-expressing lentiviruses to the mouse hippocampus ca1 region and assessment of the behavioral outcome

MicroRNAs (miRNA) er små regulatoriske enkeltstrengede RNA-molekyler omkring 22 nukleotider lange, der kan hver maalbestemt talrige mRNA-transkripter og dim en hel genekspression pathway ved at inducere ødelæggelse og / eller inhibere translation af disse mål. Flere miRNA spiller centrale roller i at opretholde neuronal struktur og funktion samt i højere niveau hjernefunktioner og metoder søges til at manipulere deres niveau for at udforske disse funktioner. Her præsenterer vi en direkte in vivo metode til at undersøge de kognitive konsekvenser af tvungne miRNA overskydende i mus ved stereotaktisk injektion af miRNA-kodning viruspartikler. Konkret den nuværende protokol indebærer injektion i hippocampus CA1 regionen, hvilket bidrager til pattedyr hukommelse konsolidering læring og stress reaktioner, og tilbyder en bekvem injektionsstedet. Koordinaterne måles efter musen bregma og virus perfusion er digitalt styret og holdt meget langsom.Efter injektion, er kirurgi såret forsegles og dyrene komme sig. Lentivirus koder lyddæmpere af de tilsvarende mRNA mål tjener at indblande den specifikke miRNA / target interaktion ansvarlig for den observerede effekt, med naive mus, mus injiceret med saltvand og mus injiceret med &quot;tomme&quot; lentivirusinfektioner vektorer som kontrolgruppe. En måned efter injektion, dyrene undersøgt i Morris Water Maze (MWM) til vurdering af deres navigation indlæring og hukommelse evner. Den MWM er en rund tank fyldt med farvet vand med en lille platform neddykket 1 cm under vandoverfladen. Steady visuelle referencer omkring tanken tillader rumlig navigation (lyd og jordens magnetfelt kan også hjælpe dyrene i at navigere). Video kamera overvågning muliggør måling af rute svømme og tid til at finde og beløbe platformen. Musen er først lært, at montere den skjulte platform tilbyder en flugt fra tvungen svømning, det er derefter testet for at bruge denne flugt og ftD KK, er platformen fjernes og probe test undersøger om musen husker sin tidligere placering. Gentagne tests over flere dage i træk fremhæve forbedret ydeevne af testede mus ved kortere ventetid til at finde og montere platformen, og som flere direkte ruter for at nå platformen eller dens placering. Manglende vise denne forbedring repræsenterer svækket indlæring og hukommelse og / eller angst, som derefter kan testes specifikt (fx i forhøjede pluslabyrint). Denne fremgangsmåde gør det muligt validering af specifikke miRNA og målrette udskrifter i den undersøgte kognitive og / eller stress-relaterede processer.

Injektion af 0,5 pi lentivirus i CA1-regionen i muse-hippocampus, med strømningshastigheden anført i protokollen sektion giver en inficeret sfære på omkring 1 mm i rostralt-caudale akse, og omkring 0,5 mm i den mediale-laterale og anterior -posterior akser (figur 3). <img alt="Figur 1" src="/files/ftp_upload/50170/50170fig1.jpg" /> Figur 1. Bregma punkt og injektionsapparatet. (A) Vist er et skematisk kort over ...

Watch this Scientific Journal Video about Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome Video at JoVE.com

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome Video (Video) | JoVE

MicroRNA har betydelige roller i hjernens struktur og funktion. Her beskriver vi en metode til at håndhæve hippocampus miRNA overekspression hjælp stereotaktisk injektion af en manipuleret miRNA-udtrykkende lentivirus. Denne fremgangsmåde kan fungere som en relativt hurtig måde at vurdere<em&gt; In vivo</em&gt; Effekter af overudtrykt miRNA i bestemte områder af hjernen.

Stereotaktisk Injektion af MicroRNA-udtrykkende lentivira til Mouse Hippocampus CA1 Region og vurdering af de Behavioral Outcome

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

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Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

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