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

Summary

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

Abstract

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Introduction

The two most reliable methods of identifying radiation-induced inter-chromosomal stable aberrations are multiple fluorescence in situ hybridization (mFISH), which allows the painting of two or more chromosomes simultaneously, and spectral karyotyping (SKY), which imparts a distinct color to each homologous chromosome pair in the genome. Unlike unstable aberrations, stable aberrations are persistent in nature and may be propagated for several generations in irradiated populations¹, and are regarded as critical molecular "signatures" of radiation-induced cytogenetic lesions². Studies by various groups have shown that stable aberrations are....

Protocol

All animal studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal protocol was approved by the Institutional Animal Care and Use Committee of the University of Arkansas for Medical Sciences. All animals were housed under standard air-conditioned animal facility at 20 ± 2 °C with 10 - 15 hourly cycles of fresh air and free access to standard rodent food and water. Upon arrival, the mice were held in quarantine for 1 week and provided certified rodent chow.

1.Radiation Exposure and In Vivo Arrest of Metapha....

Results

Total body irradiation induces numerous chromosomal aberrations in the bone marrow cells of irradiated mice. The current protocol is optimized for in vivo mitotic arrest of bone marrow cells after radiation exposure, harvest of bone marrow cells from the hind legs of irradiated mice, isolation of bone marrow mononuclear cells by density gradient centrifugation, preparation of metaphase cell spreads, and subsequent detection of radiation-induced stable chromosomal aberrations by m.......

Discussion

Several critical steps determine the success of mFISH and SKY. The first and most critical step is to optimize the colchicine treatment for in vivo mitotic arrest of the bone marrow mononuclear cells. The colchicine concentration and treatment time individually or in concert determine the mitotic index as well as chromosome condensation-two important prerequisites for effective chromosome painting. A high colchicine concentration or longer treatment time leads to highly condensed chromosomes, incompatible for pr.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Formamide	Sigma-Aldrich	221198-100ML
SSC Buffer 20× Concentrate	Sigma-Aldrich	S6639-1L
SKY Laboratory Reagent for Mouse	Applied Spectral Imaging	FPRPR0030/M40
CAD - Concentrated Antibody Detection Kit	Applied Spectral Imaging	FPRPR0033
Single Paints Customized - 3 Colors; Mouse chromosome 1: Red, Mouse chromosome 2: Green, Mouse chromosome 3: Aqua	Applied Spectral Imaging	FPRPR0182/10
Glass coverslips	Fisher Scientific	12-545B
Tween 20	Fisher Scientific	BP337-100
Hydrochloric acid, 37%, Acros Organics	Fisher Scientific	AC45055-0025
Fisherbrand Glass Staining Dishes  with Screw Cap	Fisher Scientific	08-816
KaryoMAX Potassium Chloride Solution	Life Technologies	10575-090
Fisherbrand Superfrost Plus Microscope Slides	Fisher Scientific	12-550-15
Colcemid powder	Fisher Scientific	50-464-757
Histopaque-1083	Sigma-Aldrich	10831
Shepherd Mark I, model 25 137Cs irradiator	J. L. Shepherd & Associates	Model 484B
Syringe 1 mL	BD Biosciences	647911
Ethyl Alcohol, 200 Proof	Fisher Scientific	MEX02761
PBS, (1x PBS Liq.), w/o Calcium and Magnesium	Fisher Scientific	ICN1860454
Fetal Bovine Serum	Fisher Scientific	10-437-010
Methanol	Fisher Scientific	A454SK-4
Glacial acetic acid	Fisher Scientific	AC295320010
Zeiss Microscope	Zeiss	AXIO Imager.Z2