Detectie van Inter-chromosomale Stable aberraties door Multiple Fluorescentie In Situ Hybridisatie (mFISH) en Spectral Karyotypering (SKY) in bestraalde muizen

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

Alle dierlijke studies werden uitgevoerd in strikte overeenstemming met de aanbevelingen in de Gids voor de Zorg en gebruik van proefdieren van de National Institutes of Health uitgevoerd. Het dier protocol werd goedgekeurd door de Institutional Animal Care en gebruik Comite van de Universiteit van Arkansas voor Medische Wetenschappen. Alle dieren werden gehuisvest onder standaard voorzien van airconditioning, dier faciliteit bij 20 ± 2 ° C met 10 - 15 per uur cycli van frisse lucht en gratis toegang tot standaard knaagdier voedsel en water. Bij aankomst werden de muizen in quarantaine gehouden voor 1 week en op voorwaarde dat gecertificeerd knaagdieren chow.

Results

Total body bestraling induceert talrijke chromosomale afwijkingen in de beenmergcellen van bestraalde muizen. Het huidige protocol is geoptimaliseerd voor in vivo mitose van beenmergcellen na blootstelling aan straling, oogst van beenmergcellen van de achterpoten van bestraalde muizen, de isolatie van beenmerg mononucleaire cellen door dichtheidsgradiënt centrifugatie, bereiding van metafase cel smeersels, en daaropvolgende detectie van straling veroorzaakte stabiele chromosomal.......

Discussion

Verschillende kritische stappen bepalen het succes van mFISH en SKY. De eerste en meest kritische stap is het colchicine behandeling in vivo mitose van het beenmerg mononucleaire cellen te optimaliseren. De colchicine concentratie en de duur van de behandeling individueel of in onderling overleg bepalen de mitotische index evenals chromosoom condensatie-twee belangrijke voorwaarden voor een effectieve chromosoom schilderij. Een hoge concentratie colchicine of langere behandeltijd leidt tot zeer gecondenseerde c.......

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