In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation

Nereo Kalebic; Barbara Langen; Jussi Helppi; Hiroshi Kawasaki; Wieland  B. Huttner

doi:10.3791/61171

In Vivo Targeting von neuronalen Vorläuferzellen in Ferret Neocortex durch Utero Elektroporation

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Neuroscience

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JoVE Journal Neuroscience

In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation

In Vivo Targeting von neuronalen Vorläuferzellen in Ferret Neocortex durch Utero Elektroporation

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07:03 min

May 6, 2020

DOI: 10.3791/61171-v

Nereo Kalebic^1,2, Barbara Langen^1,3, Jussi Helppi¹, Hiroshi Kawasaki⁴, Wieland B. Huttner¹

¹Max Planck Institute of Molecular Cell Biology and Genetics, ²Human Technopole, ³Landesdirektion Sachsen, ⁴Department of Medical Neuroscience, Graduate School of Medical Sciences,Kanazawa University

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Overview

This study presents a protocol for genetic manipulation in the embryonic ferret brain using in utero electroporation. The method focuses on targeting neural progenitor cells in vivo within the developing neocortex, providing insights into brain development mechanisms.

Key Study Components

Area of Science

Neuroscience
Developmental Biology
Genetic Manipulation

Background

In utero electroporation allows precision targeting of neural stem cells.
The ferret is a key model for studying neocortex expansion and folding.
This method enhances understanding of the developmental processes in the brain.
Previous studies have explored evolutionary gene functions using this approach.

Purpose of Study

To develop a reliable method for genetic manipulation in the embryonic ferret brain.
To investigate the roles of specific genes in neocortical development.
To understand the expansion and folding mechanisms of the neocortex.

Methods Used

In vivo electroporation is performed on embryonic ferrets.
DNA solution is injected into the cerebral ventricles of 33-day-old embryos.
Five electrical pulses are applied to facilitate gene expression at targeted sites.
Key steps include anesthesia, incision, and careful administration of the DNA solution.
Following electroporation, various molecular markers are used to analyze targeted cells.

Main Results

Postnatal analysis shows targeted cells differentiate into neurons and glia.
Gene expression in progenitor cells can be tracked using transcription factor markers.
Significant findings include the identification of key genes related to neocortex expansion.

Conclusions

This method opens avenues for studying gene functions in brain development.
Findings have implications for understanding evolution of the human neocortex.
The electroporation technique enables targeted manipulation of gene expression during critical developmental phases.

Frequently Asked Questions

What are the advantages of using ferrets in this study?

Ferrets provide a valuable model for studying neocortex development due to their similar developmental progression to humans.

How is the genetic manipulation implemented?

Performing in utero electroporation allows for DNA delivery specifically to neural progenitor cells, enhancing targeted studies.

What types of outcomes are obtained from this method?

The method yields vital insights into cell differentiation, gene expression, and the structural development of the neocortex.

Can this technique be adapted for other species?

Yes, while this study focuses on ferrets, in utero electroporation can be adapted for other developing mammals.

What are the key limitations of this protocol?

The procedure requires precise execution and careful monitoring of the embryos to ensure successful outcomes and minimize risks.

How can the results of this study contribute to understanding brain disorders?

By manipulating specific genes, researchers can identify pathways involved in developmental disorders and potential therapies.

What is the timeline for observing effects after electroporation?

Effects can be analyzed within days of electroporation, with significant observations noted from postnatal day zero to day 16.

Hier wird ein Protokoll zur genetischen Manipulation im embryonalen Frettchenhirn unter Verwendung der Utero-Elektroporation vorgestellt. Diese Methode ermöglicht die Ausrichtung von neuronalen Vorläuferzellen im Neocortex in vivo.

Dieses Protokoll ermöglicht es uns, eine genetische Manipulation in vivo in einem wichtigen Tiermodell durchzuführen, das es uns ermöglicht, die Ausdehnung und Faltung eines sich entwickelnden Neocortex zu untersuchen. Der Hauptvorteil dieser Methode besteht darin, eine hohe räumliche und zeitliche Spezifität zu verwenden, wenn sie auf jene neuronalen Stammzellen abzielt, die der Schlüsselzelltyp für die Entwicklung des Gehirns sind. Ziehen Sie vor Beginn des Verfahrens Glaskapillaren an einem Mikropipettenzieher und schneiden Sie mit Zangen den distalen Teil der Kapillare ab, um den Durchmesser der Kapillarspitze anzupassen.

Am embryonalen Tag 33 die entsprechende DNA-Konzentration in PBS hinzufügen, ergänzt mit 0,1%Fast Green mit sanftem Mischen, und legen Sie ein 3-stündiges, anästhesiertes, schwangeres weibliches Frettchen auf einen Operationstisch mit einem Heatpad. Bestätigen Sie den entsprechenden Sedierungsgrad, indem Sie die periokuläre Haut berühren und die Haut zwischen dem zweiten und dritten oder dritten und vierten Zehen beider Hinterbeine kneifen. Isofluran kann Nebenwirkungen verursachen, einschließlich Übelkeit und Schwindel.

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