Påvisning av Inter-kromosom Stabil Avvik ved Multiple fluorescens<em> In Situ</em> Hybridisering (mFISH) og Spectral karyotypering (SKY) i Bestrålet Mus

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 associated with the pathogenesis and development of a number of diseases, including cancer³. Before the era of chromosome painting (also referred as molecular cytogenetics), the conventional G-banding technique was the only method for detecting stable chromosomal aberrations. However, chromosome banding is a challenge to cytogeneticists because the resolution is limited, reproducibility is uncertain, it is a labor-intensive procedure, and it requires highly skilled and experienced cytogeneticists for reliable data interpretation⁴. Moreover, the classic banding technique does not allow detection of complex chromosomal rearrangements, which involve the interaction of three or more breaks distributed among two or more chromosomes, a common outcome of radiation damage. Complex aberrations may persist in individuals many years after radiation exposure, making them useful for retrospective biodosimetry⁵. Therefore, an alternate approach was required to overcome the limitations of conventional banding techniques to detect stable chromosomal rearrangements.

In the late 1960s, the pioneering work of Gall and Pardue (1969) on molecular hybridization using nucleic acid probes labeled with radioactive material commenced a new era in the field of cytogenetics, which allowed detection of a specific DNA sequence on chromosomes⁶. However, the use of radioactive probes for molecular hybridization had several drawbacks: radioactive probes are relatively unstable, probe activity depends on radioactive decay of the isotope used, hybridization takes a longer time, the resolution is limited, the probes are relatively costly, and the radioactive materials are a health hazardous. Thus, it became necessary to develop and design non-radioactive probes. The introduction of fluorescent tagged nucleic acid probes in the 1980s and 1990s overcame the limitations of radioactive probes and greatly enhanced the safety, sensitivity, and specificity of the hybridization technique^7-10. Fluorescent probes give rise to extremely bright signals when observed under fluorescence microscopes equipped with the appropriate excitation and emission filters. Any loss, gain, or rearrangement of fluorescent labeled chromosome/s or a part of the chromosome is easily identifiable with this FISH technique.

Analysis of chromosomal aberrations by FISH painting has led to marked progress in cytogenetic research over the years. Designing fluorescent labeled probes for specific applications ranging from locus-specific probes to whole-chromosome painting probes has advanced the field significantly; this has also facilitated the detection of submicroscopic (“cryptic”) rearrangement, which was not possible by conventional chromosome banding. Chromosome painting by mFISH and SKY have proven to be valuable tools for the identification of simple and complex inter-chromosomal rearrangements. The basic principles for both techniques are similar, but the method of detection and discrimination of fluorescent signal after in situ hybridization and the process of image acquisition are different. In mFISH, separate images of each of the four fluorochromes are captured by using narrow bandpass microscope filters; dedicated software is then used to combine the images. While in SKY, image acquirement is based on a combination of epifluorescence microscopy, charge-coupled device imaging, and Fourier spectroscopy, which allows the measurement of the entire emission spectrum with a single exposure at all image points. In both mFISH and SKY, monochrome images are captured independently, then merged, and finally, unique pseudo-colors are assigned to the chromosomes in monochromatic images based on the specific dye attached to each fluorochrome probe.

The contribution of mFISH and SKY analysis in the radiation biology field is remarkable, particularly for retrospective dose estimation of human exposure to IR (radiation biodosimetry)^11-14, radiation carcinogenesis risk assessment¹⁵, as well as detection and risk estimation of radiotherapy-related secondary cancer¹⁶. A recent study on mice has shown that a FISH-based chromosome painting technique is also an important tool for evaluating the efficacy of radiation countermeasure¹⁷. In the present study, the effect of total body radiation exposure on the induction of stable chromosomal aberrations in the bone marrow cells of mice has been demonstrated using mFISH and SKY techniques.

Protocol

Alle dyrestudier ble utført i henhold til anbefalingene i Guide for omsorg og bruk av forsøksdyr av National Institutes of Health. Dyret protokollen ble godkjent av Institutional Animal Care og bruk komité ved University of Arkansas for Medical Sciences. Alle dyr ble holdt under standard aircondition Dyreavdelingen ved 20 ± 2 ° C med 10 – 15 time sykluser av frisk luft og fri tilgang til standard gnager mat og vann. Ved ankomst, ble musene holdt i karantene i en uke og ga sertifisert gnager chow. <p class="jove…

Representative Results

Helkroppsbestråling induserer mange kromosomavvik i benmargceller av bestrålte mus. Den nåværende protokollen er optimalisert for in vivo mitotisk arrest av benmargceller etter stråling, høsting av benmargceller fra bakbena bestrålte mus, isolering av benmarg mononukleære celler ved densitetsgradientsentrifugering, utarbeidelse av metacelle sprer seg, og etterfølgende deteksjon av stråling-indusert stabile kromosomavvik ved mFISH og SKY teknikker. Kromosom maling ved h…

Discussion

Flere kritiske trinn avgjør suksessen mFISH og SKY. Den første og mest kritiske trinnet er å optimalisere kolkisinbehandlingen for in vivo mitotisk arrest av benmarg mononukleære celler. Den kolkisin konsentrasjon og behandlingstiden individuelt eller i konsert bestemme mitotisk indeks samt kromosomkondens to viktige forutsetninger for effektiv kromosom maleri. En høy kolkisin konsentrasjon eller lengre behandlingstiden fører til kondenserte kromosomer, inkompatible for riktig denaturering og hybridiserin…

Materials

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