Name: in ovo electroporation in chick midbrain for studying gene function in dopaminergic neuron development
Uploaded: 2012-08-03T13:00:00
Description: Watch this Scientific Journal Video about In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development at JoVE.com

Vi takker Dr. Takahiko Matsuda til tilvejebringelse af pCAG-mCherry konstruktion. YCM understøttes af en karriereudvikling Award fra Schweppe Foundation og en bevilling fra Whitehall Foundation.

1. Forsyning Forberedelse (ikke video) <ol><li> Fremstille en frisk 1X fortynding af Penicillin / Streptomycin (Pen / Strep) i 1X PBS. Ikke mere end 50 ml, er nødvendig. Filter med en 0,22 um sprøjtefilter. </li><li> Forberede injektion konstruktioner ved at kombinere ønskede plasmidkonstruktioner. Vi sætter alle konstruktioner i pCAG-Flag-vektoren med passende støkiometri til et slutvolumen på ~ 10 uL. Desuden bør pCAG-GFP 14 eller pCAG-mCherry lægges til sporing af elektroporation effektivitet og ...

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In ovo elektroporering af embryoniske kylling midthjernen giver en billig og hurtig alternativ til generering af transgene eller knockout-dyr at udføre in vivo undersøgelse af genfunktioner i midthjerne-dopaminerge neuron udvikling. Ved hjælp af korte 2 mm lange L-formet platinelektroder med embryoniske midthjernen-specifikt DNA-injektion er nøglen til at opnå effektiv ekspression af genet af interesse i midthjerne-dopaminerge neuron progenitorer. Hertil kommer, er ved hjælp af korrekte HH Etape ...

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in ovo electroporation in chick midbrain for studying gene function in dopaminergic neuron development

Dopaminerge neuroner beliggende i den ventrale midthjernen styrer bevægelser, følelsesmæssig adfærd og belønning mekanismer 1-3. Dysfunktion af ventrale midthjerne-dopaminerge neuroner er involveret i Parkinsons sygdom, skizofreni, depression og demens 1-5. Således kunne studere reguleringen af ​​midthjernen dopaminerg neuron differentiering ikke kun give vigtig indsigt i mekanismer, der regulerer midthjernen udvikling og neurale stamceller skæbne specifikation, men også hjælpe med at udvikle nye terapeutiske strategier for behandling af forskellige mennesker neurologiske lidelser. Dopaminerge neuroner skelne fra neurale stamceller, der beklæder ventrikulære zone af embryonisk ventral midthjernen. Udviklingen af neurale stamceller er styret af genekspression programmer 6,7. Her rapporterer vi teknikker anvender elektroporation til at udtrykke gener specifikt i midthjernen af ​​Hamburger Hamilton (HH) scene 11 (thirteen somitter, 42 timer) kyllingeembryoer 8,9. Den eksterne Udviklingen af kyllingeembryoer giver mulighed for bekvem eksperimentelle manipulationer på specifikke fosterstadiet, med de følgevirkninger afgøres på et senere udviklingsmæssige tidspunkter 10-13. Chick embryoniske neurale rør tidligere end HH trin 13 (nitten somitter, 48 timer) består af multipotente neurale stamceller som er i stand til at differentiere til forskellige celletyper i nervesystemet. Den pCAG vektor, som indeholder både en CMV-promotor og en kylling β-actin-enhancer, tillader robust ekspression af Flag-eller andre epitop-mærkede konstruktioner i embryoniske kyllinge neurale rør 14. I denne rapport, lægger vi vægt på særlige foranstaltninger for at opnå regionalt begrænset genekspression i embryonale midterhjernedopaminerge dopaminerge neuron stamceller, herunder hvordan at injicere DNA-konstruktionerne specifikt til den embryonale midthjernen regionen og hvordan man kan lokalisere elektroporation med små skræddersyede elektroder. Analyse cHick midthjernen på senere stadier tilvejebringer et fremragende in vivo for plasmidvektoren-medieret gain-af-funktion og tab af funktion undersøgelser af midterhjernen udvikling. Modifikation af det eksperimentelle system kan strække assayet til andre dele af nervesystemet til udførelse skæbne kortlægningsanalyse og til undersøgelse af reguleringen af ​​genekspression.

Watch this Scientific Journal Video about In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development at JoVE.com

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

At vurdere funktionen og reguleringen af ​​gener under udviklingen af ​​midthjerne-dopaminerge neuroner, beskriver vi en fremgangsmåde, der involverer<em&gt; In ovo</em&gt; Elektroporering af plasmid-DNA-konstruktioner i embryoniske kyllinge ventrale midthjerne-dopaminerge neuron progenitorer. Denne teknik kan anvendes til at opnå effektiv ekspression af gener af interesse at undersøge forskellige aspekter af midterhjerne udvikling og dopaminerg neuron differentiering.

I ovo Elektroporation i Chick midthjernen for at studere genfunktion i dopaminerg Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

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In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

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