Name: ex utero electroporation and organotypic slice culture of mouse hippocampal tissue
Uploaded: 2015-03-04T15:00:00
Description: Watch this Scientific Journal Video about Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue at JoVE.com

This work was supported by grants from the Deutsche Forschungsgemeinschaft to SB (BR-2215; SFB 497/A9).

BEMÆRK: Alle dyreforsøg blev udført i henhold til den tyske lov og blev godkendt af offentlige kontorer i Tübingen. 1. Fremstilling af mikropipetter, løsninger og membraner <ol><li> Fremstilling af Mikropipetter <ol><li> Træk glas mikropipetter ved hjælp af en mikropipette aftrækker med følgende program: Varme: 540, Pull: 125, Hastighed: 20 og Forsinkelse: 140. De nålelængde udgør 5,5 cm. </li><li> Koniske nåle bruger microgrinder at opnå en passende spids størrelse på 4 mm...

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Hippocampus har en vigtig funktion i indlæring og hukommelse. Den tandede gyrus er også en af ​​to hjerneområder, hvor neurogenese optræder ikke kun under udvikling, men også gennem hele voksenlivet. Fødselsdepression og voksne hippocampus neurogenese provenu på samme måde involverer mange lighedspunkter. Definition de regulatoriske mekanismer disse faktorer vil være meget nyttigt at forstå neurodegenerative sygdomme, hvilket igen vil føre til nye behandlingsformer og forebyggende foranstaltninger. For at...

Hippocampus spiller en vigtig rolle i hukommelse og indlæring samt følelsesmæssig adfærd. En funktion vigtigste består i konsolideringen af ​​korttidshukommelsen i langtidshukommelsen, som kræver høj plasticitet af nervesystemet. Gyrus dentatus i hippocampus fungerer som den primære gateway for input information og er også en af to områder af hjernen med igangværende neurogenese gennem hele voksenlivet 1,2. Udviklingen af hippocampus struktur sker under sen embryogenese og især i de første 3 til 4 uger efter fødslen 3. I den tidlige udvikling af gyrus dentatus en stamcelle pool etableres nødvendig for postnatal samt voksen neurogenese 4. Udvikling neuroner passerer gennem forskellige stadier, fra stamceller gennem flere stadier af stamceller til umodne og endelig den modne neuron under postnatal samt voksen neurogenese. På forskellige stadier af neurogenese udtryk forspecifikke gener er nødvendig for at gøre det muligt for modning og integration af nye neuroner i hippocampus kredsløb 5,6. Ved hjælp af musen genetik og fænotype-analyse ved immunohistokemi samt molekylære metoder tilladt definere ekspressionsmønster og funktion af mange af disse gener. Desuden microarray analyse samt kromatin immunoprecipitation (chip) givet oplysninger om potentielle direkte og indirekte målgener 7,8. Men der er stadig mange åbne spørgsmål vedrørende de regulatoriske mekanismer hippocampal udvikling, især udviklingen af ​​gyrus dentatus. For at få yderligere indsigt hvordan specifikke gener reguleres et system kræves tillader manipulation af et lille antal celler ved ned- eller opregulering af genet af interesse og / eller dets målgener og følge deres skæbne under udvikling. I livmoderen elektroporation af shRNAs, cDNA af gener af interesse eller Cre recombinaSE giver et sådant værktøj. For at sikre tilstedeværelsen af ​​det ønskede DNA eller små RNA ekspressionsplasmider bør anvendes til elektroporation. Denne fremgangsmåde er meget gennemført med succes i at studere cortical udvikling 9,10, men er en mere udfordrende tilgang undersøge udviklingen af gyrus dentatus grund af placeringen af de hippocampale strukturer i dybere hjerne lag. Ex utero elektroporation efterfulgt af organotypisk skive kultur er en metode til at omgå dette problem 11,12. I modsætning til in utero elektroporation ikke hele embryo, men kun hovedet anvendes tillader derfor at placere elektroderne i en mere gunstig måde at lede shRNA / DNA mod hippocampus og gyrus dentatus. Vores gruppe med held ex utero elektroporation at studere rollen af transkriptionsfaktoren Bcl11b under gyrus dentatus udvikling 8. Bcl11b har en dobbelt rolle i tandede gyrus udvikling ved regulating progenitor celleproliferation samt differentiering som blev påvist ved immunhistokemi. For yderligere at definere en mekanisme til Bcl11b deltagelse i disse processer, er protokoller til Polleux gruppe 11,12 justeres for at studere den tandede gyrus som beskrevet nedenfor i protokollen sektion. I en første tilgang til spørgsmålet var rettet, om Bcl11b regulerer neuronal celledifferentiering celle selvstændigt. En anden metode undersøgt desmoplakin, et direkte mål gen Bcl11b, er tilstrækkelig til at redde Bcl11b fænotype.

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ex utero electroporation and organotypic slice culture of mouse hippocampal tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ablation af transkriptionsfaktoren Bcl11b forårsager svækkelse af progenitor celleproliferation og neuronal differentiering resulterer i en reduceret gyrus dentatus størrelse og celleantal. Desuden mutant neuroner ikke at blive integreret i hippocampus kredsløb forårsager indlæring og hukommelse værdiforringelse 8. For at besvare spørgsmål vedrørende reguleringsmekanisme (r) Bcl11b i disse processer ex utero elektroporation blev ansat. Behandle spørgsmålet om,...

Watch this Scientific Journal Video about Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue at JoVE.com

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Ex Utero Elektroporering og organotypisk Slice kultur Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Research

JoVE Journal

Developmental Biology

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

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Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific ...

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Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

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The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

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Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

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The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...