Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Patrick Bovio; Deborah Roidl; Stefanie Heidrich; Tanja Vogel; Henriette Franz

doi:10.3791/56631

בידוד, טיפוח של אבות עצבית ואחריו Immunoprecipitation-כרומטין של היסטון 3 ליזין 79 Dimethylation ...

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JoVE Journal Neuroscience

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

בידוד, טיפוח של אבות עצבית ואחריו Immunoprecipitation-כרומטין של היסטון 3 ליזין 79 Dimethylation מארק

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10:09 min

January 26, 2018

DOI: 10.3791/56631-v

Patrick Bovio^1,2, Deborah Roidl¹, Stefanie Heidrich¹, Tanja Vogel¹, Henriette Franz¹

¹Institute for Anatomy and Cell Biology, Department of Molecular Embryology, Faculty of Medicine,University of Freiburg, ²Faculty of Biology,University of Freiburg

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Overview

This study presents a reproducible method for isolating and culturing neural progenitor cells from embryonic and postnatal brain tissue. The focus is on analyzing epigenetic modifications, specifically the histone mark H3K79me2, during brain development within the cerebral cortex and cerebellum.

Key Study Components

Area of Science

Neuroscience
Epigenetics
Cell Culture Techniques

Background

Neural progenitor cells are crucial for brain development.
Histone modifications play a significant role in gene expression.
The study investigates the specific impact of H3K79 methylation.
Understanding these mechanisms can provide insights into cortical and cerebellar layering.

Purpose of Study

To analyze epigenetic changes during brain development.
To explore the effects of histone modifications on neural differentiation.
To develop a reliable protocol for ChIP analyses of specific histone marks.

Methods Used

Cell culture techniques utilizing isolated neural progenitor cells.
Focus on embryonic and postnatal brain tissues, specifically cerebellar cells.
Instructions for tissue digestion, cell isolation, and histone extraction.
Detailed steps for Chromatin Immunoprecipitation (ChIP) to analyze histone modifications.

Main Results

The method successfully isolates and cultures cerebellar neural progenitor cells.
ChIP protocols can analyze H3K79me2 levels in various development stages.
Findings indicate a significant relationship between histone modifications and neuronal development.
Validations confirm the effectiveness and reproducibility of the technique.

Conclusions

This study demonstrates an effective methodology for exploring epigenetic regulation in neural development.
The approach enables detailed investigation into how histone modifications influence the neurodevelopmental process.
Insights gained can contribute to understanding neuronal mechanisms and potentially implicate disease models.

Frequently Asked Questions

What are the advantages of this cell culture method?

This method allows for the study of neural progenitor cells in a controlled environment, making it reproducible and efficient for epigenetic analysis.

How are the neural progenitor cells isolated?

The cells are isolated from cerebellar tissue by digestion, centrifugation, and trituration, ensuring a high yield of viable progenitor cells.

What type of data can be obtained from this method?

Researchers can obtain molecular readouts related to histone modifications and assess the impacts on cellular behavior and development.

How can this method be adapted for other brain regions?

The protocol can be modified for different brain regions by altering the source tissue and optimizing the digestion and culture conditions accordingly.

Are there any limitations to this technique?

Key limitations include the potential variability in cell yield and the requirement for specific tissue sources, which may not always be available.

What implications does this study have for neurobiology?

The findings enhance the understanding of epigenetic regulation in brain development and may inform future research on neurodevelopmental disorders.

What steps are involved in the ChIP process?

The ChIP process includes cell lysis, chromatin shearing, immunoprecipitation with specific antibodies, and subsequent analysis to assess histone modification levels.

אנו מציגים שיטה לשחזור ויעילים כדי לבודד את תרבות ובתאים עצבית של רקמת מוח עובריים ופוסט עבור immunoprecipitation כרומטין (ChIP) של היסטון 3 ליזין 79 dimethylation (H3K79me2) - סימן היסטון ממוקם במרחק תחום הכדוריים של היסטון 3.

המטרה הכוללת של הליך זה היא לנתח שינויים אפיגנטיים במהלך התפתחות המוח, במיוחד בקליפת המוח ובמוח הקטן. ניתן להשתמש בשיטה זו כדי לענות על שאלות מפתח בתחום הנוירוביולוגיה, כגון כיצד שינויים בהיסטון כמו מתילציה של H3K79 יכולים להשפיע על התפתחות שכבות קליפת המוח והמוח. היתרון העיקרי של טכניקה זו הוא שאנו יכולים לחקור in vivo את השינויים בהיסטון, ספציפיים ללוקוס או בכל הגנום, באזורי מוח שונים בנקודות זמן התפתחותיות ספציפיות.

התחל עם מוחות מ-P5 עד P7 ועכברי MRI. הסר את כל קרומי המוח וכלי הדם. לאחר מכן העבירו שלושה עד חמישה מוח קטן לכל שבב לצינורות של 15 מיליליטר.

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