Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence <em>In Situ</em> Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Rupak Pathak; Igor Koturbash; Martin Hauer-Jensen

doi:10.3791/55162

Çoklu Floresan Inter-kromozomal Kararlı sapmaları Algılama In Situ Hibridizasyon (mFISH)...

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Genetics

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JoVE Journal Genetics

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Çoklu Floresan Inter-kromozomal Kararlı sapmaları Algılama In Situ Hibridizasyon (mFISH) ve ışınlanmış Farelerde Spektral Karyotipik (SKY)

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10:14 min

January 11, 2017

DOI: 10.3791/55162-v

Rupak Pathak¹, Igor Koturbash², Martin Hauer-Jensen^1,3

¹Division of Radiation Health, Department of Pharmaceutical Sciences, College of Pharmacy,University of Arkansas for Medical Sciences, ²Department of Environmental Health, Fay W. Boozman School of Public Health,University of Arkansas for Medical Sciences, ³Surgical Service,Central Arkansas Veterans Healthcare System

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Overview

This protocol outlines the application of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) to identify inter-chromosomal stable aberrations in the bone marrow cells of mice following total body irradiation. This method is significant in understanding the genetic impacts of radiation exposure.

Key Study Components

Area of Science

Radiation Biology
Cytogenetics
Molecular Biology

Background

Stable chromosomal aberrations can be induced by radiation exposure.
Bone marrow cells are critical for studying genetic damage.
Understanding these aberrations helps assess radiation risks.
mFISH and SKY are advanced techniques for visualizing genetic changes.

Purpose of Study

To observe interchromosomal stable aberrations in bone marrow cells.
To evaluate the risk of stable chromosomal damage from total body irradiation.
To provide a method for guided visualization of genetic damage.

Methods Used

Isolation of bone marrow from mouse femur and tibia.
Preparation of a single cell suspension.
Use of bone marrow mononuclear cell separation medium.
Centrifugation to collect the buffy coat for analysis.

Main Results

Identification of stable chromosomal aberrations in irradiated cells.
Demonstration of the effectiveness of mFISH and SKY techniques.
Insights into the propagation of genetic damage across cell generations.
Contribution to understanding radiation-induced genetic risks.

Conclusions

mFISH and SKY are valuable tools for studying radiation effects.
Stable aberrations can be tracked through multiple cell divisions.
This research aids in assessing the long-term impacts of radiation exposure.

Frequently Asked Questions

What is the significance of studying chromosomal aberrations?

Studying chromosomal aberrations helps assess the genetic risks associated with radiation exposure.

How are bone marrow cells isolated for this study?

Bone marrow is isolated from mouse femur and tibia, followed by creating a single cell suspension.

What techniques are used to visualize genetic damage?

Multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) are used for visualization.

What are the main findings of this research?

The research identifies stable chromosomal aberrations in irradiated bone marrow cells and demonstrates the effectiveness of the applied techniques.

How does this study contribute to radiation biology?

It provides insights into the risks of stable genetic damage from radiation exposure, which is crucial for understanding long-term effects.

Can the results of this study be applied to other organisms?

While this study focuses on mice, the methodologies may be adapted for other species in radiation research.

Mevcut protokol, toplam vücut ışınlamasına maruz kaldıktan sonra farelerin kemik iliği hücrelerindeki kromozomal stabil anormallikleri tanımlamada çoklu floresan in situ hibridizasyon (mFISH) ve spektral karyotiplemenin (SKY) yararlılığını açıklamaktadır.

Bu moleküler sitojenik yöntemin genel amacı, toplam vücut ışınlamasına maruz kalan farelerin kemik iliği hücrelerinde kromozomal stabil anormallikleri gözlemlemektir. Bu yöntem, radyasyon biyolojisi alanında, toplam vücut radyasyonuna maruz kalan farelerin kemik iliği hücrelerinde stabil kromozomal anormalliklere neden olma riski gibi kilit soruyu yanıtlamaya yardımcı olabilir. Bu tekniğin ana avantajı, farelerin kemik iliği hücrelerinde radyasyonun neden olduğu stabil genetik hasarların rehberli olarak görselleştirilmesine izin vermesidir ve bu hasarlar birçok hücre nesli boyunca çoğaltılabilir.

Fare femur ve tibia kemiklerinden kemik iliğini izole ettikten ve metin protokolüne göre tek hücreli bir süspansiyon yaptıktan sonra, hücre süspansiyonunu eşit hacimde kemik iliği mononükleer hücre ayırma ortamı üzerine dikkatlice yerleştirin. Gradyanı oda sıcaklığında 30 dakika boyunca 400 kez G'de santrifüjleyin. Ardından, kalan katmanları bozmadan buffy kaplamayı dikkatlice toplayın ve çözeltiyi yeni bir 15 mililitrelik santrifüj tüpüne aktarın.

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