Name: detection of inter-chromosomal stable aberrations by multiple fluorescence in situ hybridization (mfish) and spectral karyotyping (sky) in irradiated mice
Uploaded: 2017-01-11T15:00:00.000+00:00
Duration: 10 min 14 s
Description: Watch this Scientific Journal Video about Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice at JoVE

Diese Studie wurde von Arkansas Raum Grants Consortium und National Space Biomedical Research Institute durch National Aeronautics and Space Administration unterstützt wurde, gewährt NNX15AK32A (RP) und RE03701 (MH-J) und P20 GM109005 (MH-J) und die US Veterans Administration ( MH-J). Wir danken Christopher Fettes, Programm-Koordinator für die Abteilung für Umwelt- und Arbeitsmedizin an der University of Arkansas for Medical Sciences, für redaktionelle Unterstützung bei der Vorbereitung des Manuskripts.

Alle Tierversuche wurden in strikter Übereinstimmung mit den Empfehlungen im Leitfaden für die Pflege und Verwendung von Labortieren der National Institutes of Health durchgeführt. Das Tier Protokoll wurde von der Institutional Animal Care und Use Committee der University of Arkansas for Medical Sciences genehmigt. 15 Stundentakt von frischer Luft und den freien Zugang zu Standard-Nagerfutter und Wasser - Alle Tiere wurden mit 10 bei 20 ± 2 ° C unter Standard klimatisierten Tieranlage untergebracht. Bei der Ankunft wurden die Mäuse für 1 Woche in Quarantäne gehalten und zertifiziertes Nagerfutter zur Verfügung gestellt. 1.Radiation Belichtung und In - vivo - Arrest von Metaphasezellen <ol><li> Expose un-narkotisierten Mäusen 2 Gy Ganzkörperbestrahlung in einer Bestrahlungskammer. Während der Bestrahlung statt Mäuse in einem gut belüfteten Aufnahmekammer sicherstellen, dass sie sich nicht frei bewegen und eine einheitliche Dosis erhalten. </li><li> Inject 100 ul von 0,05% Colchicin-Lösung (colchicine Pulver Calcium und Magnesium-freiem PBS gelöst in) intraperitoneal unter Verwendung einer 25 G Nadel befestigt mit 1 ml Einwegspritze. Vermeiden Sie direkte Injektion in jedem Organ. </li><li> Lassen Sie das Tier in den Käfig für 2 h vor Knochenmarkzellernte. Beobachten Sie das Tier auf Anzeichen von Schmerzen oder Leiden. </li></ol> 2. Knochenmarkzellernte und Isolierung von Knochenmark mononukleären Zellen durch Dichtegradientenzentrifugation <ol><li> Euthanize die Maus durch CO 2 Ersticken. </li><li> Spray 70% Ethanol auf der dorsalen und ventralen Seite. </li><li> Machen Sie eine 2 cm lange Inzision auf der Bauchhaut mit einer scharfen Schere. Fassen Sie die Haut auf beiden Seiten des Einschnitts mit stumpfen Pinzette und ziehen Sie vorsichtig die Bauchmuskeln zu öffnen. </li><li> Schneiden Sie sowohl die Hinterbeine mit einer Schere und sofort legen Sie sie in vorgekühlte PBS mit 4% FBS. </li><li> sorgfältig reinigen, alle an den hinteren Gliedmaßen mit einem scharfen Rasierklinge und Baumwolle Mullbinde ansetzenden Muskeln. </li><li> Trennen Sie die Tibia vom Femur. Schneiden Sie die beiden Spitzen jedes Knochen mit einer Rasierklinge. </li><li> Spülen des Inhalts des Knochenmarks mit 3 ml PBS (mit 4% FBS) unter Verwendung einer 23 G-Nadel an einer 3 ml Spritze und sammeln den Inhalt in ein 15 ml Zentrifugenröhrchen. Übergeben Sie die Zellsuspension mindestens 10 Mal durch die Nadel eine Einzelzellsuspension zu machen. </li><li> Overlay sorgfältig die Zellsuspension auf einem gleichen Volumen von Lymphozytentrennmedium (3 ml), ohne die Schnittstelle zwischen der Zellsuspension und Lymphozyten-Separationsmedium zu stören. Zentrifuge bei 400 × g für 30 min bei Raumtemperatur. </li><li> Sammeln Sie die Leukozytenmanschette vorsichtig, ohne zu stören die anderen Schichten und die Übertragung in einem neuen 15 ml Zentrifugenröhrchen. </li></ol> 3. Herstellung von Metaphase Zelle Aufstriche <ol><li> 10 mL PBS auf das Rohr die buffy coat enthält. </li><li> Zentrifuge bei 400 × g für 5 min bei Raumtemperatur. </li><li> Entfernen Sie vorsichtig den Überstand. Dann brechen ter Pellet mit leichtes Klopfen Zelle und 10 ml PBS hinzufügen. </li><li> Zentrifuge bei 400 × g für 5 min bei Raumtemperatur. </li><li> Entfernen Sie den Überstand ohne das Zellpellet zu stören, brechen das Pellet auf, und 4 ml vorgewärmten hypotone 0,075 M Kaliumchloridlösung. In hypotone Lösung tropfenweise mit sanften konstantem Schütteln. </li><li> Inkubiere Zellen für 20 min bei 37 ° C in einem Wasserbad. </li><li> Nach dem hypotonischer Behandlung, ein gleiches Volumen (4 ml) Fixiermittel (Methanol: Eisessig, 3: 1 v / v) vorsichtig in den 15 ml-Röhrchen und mischen Sie den Schlauch durch Umkehren. </li><li> Zentrifuge bei 400 xg für 5 min bei Raumtemperatur und den Überstand verwerfen. </li><li> Brechen Sie das Zellpellet mit von sanften Wirbel für ein paar Sekunden, gefolgt tippen und 3 ml Fixierungslösung tropfenweise mit konstantem Schütteln. </li><li> Inkubieren für 30 min bei Raumtemperatur und dann bei 400 xg für 5 min zentrifugieren. </li><li> Entfernen Sie den Überstand, brechen die Zellpellet mit gent uple tippen und 3 ml frisches Fixiermittel. </li><li> Zentrifuge bei 400 xg für 5 min bei Raumtemperatur, und entfernen Sie den Überstand. Brechen Sie das Zellpellet mit leichtes Klopfen auf, und fügen Sie frische 3 ml Fixiermittel. </li><li> Wiederholen Sie Schritt 3.12 zweimal. </li><li> Zentrifuge bei 400 × g für 5 min bei Raumtemperatur und Entfernen von Überstand ohne das Zellpellet zu stören. Dann fügen Sie 400 bis 600 &amp; mgr; l Fixiermittel und gründlich mischen mit Pipettieren. </li><li> Drop-30 &amp; mgr; l fixierten Zellen auf vorgereinigte und nassen Rutschen bei einem Winkel von 45 ° geneigt und lassen Sie die Folien an der Luft trocknen über Nacht vollständig. </li></ol> 4. Maus Chromosome Painting von MFISH <ol><li> Chromosome Denaturierung <ol><li> Legen Sie die Folie mit der Metaphase Zelle Aufstriche in 2 × SSC für 2 Minuten bei Raumtemperatur. </li><li> Entwässern Sie die Folie durch serielle Ethanol Waschen für jeweils 2 Min in 70%, 80% und 100%. bei 65 ° C für 60 min inkubieren. </li><li> Vorheizen 40 ml Denaturierungslösung (70% Formamid / 2x SSC; pH 7,0) auf 70 ° C (± 2 ° C) in einem Glas Coplin jar für 30 min. Denaturieren Chromosomen durch die Folie in vorgewärmten Denaturierungslösung Inkubation mit 1 - 1,5 min. </li><li> Quench rutschen sofort in eiskaltem 70% Ethanol für 2 min die Denaturierung als auch zu stoppen, zu verhindern, dass die denaturierten Chromosomen wieder Glühen. Dann Dehydratisieren mit 80% Ethanol für 2 min durch Waschen. Wiederholen des Ethanols Wäsche mit 100% Ethanol für 2 min. vollständig trocknen die Folie bei Raumtemperatur. </li></ol></li><li> Denaturierung und Hybridisierung der Sonde Mischung <ol><li> Kurz die Sondenmischung vom Hersteller geliefert, Zentrifuge, übertragen 10 &amp; mgr; l Probenmischung in einen 500 &amp; mgr; l Kappe Zentrifugenröhrchen schnappen, und für 7 min bei 80 ° C (± 2 ° C) in einem Wasserbad durch Inkubation denaturieren. </li><li> Platzieren Sie die Zentrifugenröhrchen mit Probenmischung in einem Wasserbad bei 37 ° C für 10 min. </li><li> Tragen Sie die denaturierte Sonde Mischung auf den Objektträger mit denaturierten chromosomes. </li><li> Deckel vorsichtig den Bereich mit einem 18 mm x 18 mm Deckglas und beseitigen alle sichtbaren Luftblasen durch sehr sanft das Deckglas drücken gleiten. Dichten Sie alle vier Seiten des Deckglases mit Gummizement und Inkubation für 12 h bis 16 h im Dunkeln bei 37 ° C in einer befeuchteten Kammer für die Hybridisierung. </li></ol></li><li> Beitrag Hybridization Waschen und Nachweis <ol><li> Entfernen Sie vorsichtig den Gummizement und das Deckglas. </li><li> Ort Schieber in vorgewärmten 0,4x SSC bei 74 ° C (± 2 ° C) für 5 min. </li><li> Wasch slide in Waschlösung II (4x SSC / 0,1% Tween-20) für 2 min. </li><li> Legen Sie 20 &amp; mgr; l anti-fade Eindeckmedium mit DAPI counterstain (1,5 ug / ml) geben und mit einem Deckglas. Drücken Sie vorsichtig auf Deckglas mit Labor Gewebe, um Luftblasen und überschüssige Montagelösung zu entfernen. Seal die Ränder des Deckglases mit Nagellack. </li><li> Blick gleitet ein Fluoreszenzmikroskop mit geeigneten Filtern. </li></ol></li&gt; </ol> 5. Spektrale Karyotypisierung (SKY) von Maus-Chromosome <ol><li> Chromosome und Probe Denaturierung <ol><li> Äquilibrieren Folie in 2x SSC bei Raumtemperatur für 2 Minuten, ohne Schütteln. </li><li> Dehydratisieren Folie in einer Ethanolreihe (70%, 80% und 100% Ethanol) für 2 min jeweils bei Raumtemperatur. Der Luft trocknen die Folie, um das Ethanol vollständig zu entfernen. </li><li> Warme 40 ml Denaturierungslösung (70% Formamid / 2 × SSC, pH 7,0) in einem Glas Glasküvette 30 min. </li><li> Inkubiere Objektträger in vorgewärmten Denaturierungslösung für 1 bis 1,5 min. </li><li> Tauchen Sie sofort das Abgleiten in eiskaltem 70% Ethanol für 2 min die Denaturierung als auch zu stoppen, wie auf die denaturierten Chromosomen wieder Glühen verhindern. </li><li> Entwässern die Folie, indem sie in 80% Ethanol für 2 min und dann in 100% Ethanol für 2 min bei Raumtemperatur setzt. </li><li> Denaturieren die SKY-Sonde (Vial # 1 durch Hersteller mitgeliefert) in einem Wasserbad auf 80 ° C (± 2 ° C)7 min, und legen Sie dann sofort in ein anderes Wasserbad für 10 min auf 37 ° C eingestellt. </li></ol></li><li> Sondenhybridisierung <ol><li> Fügen Sie 10 ul der denaturierten SKY-Sonde auf die denaturierten Chromosomen. </li><li> eine 18 mm x 18 mm Deckglas auf den SKY-Sonde vorsichtig platzieren, so dass keine Luftblasen unter dem Deckglas gefangen sind. </li><li> Seal die Ränder des Deckglases mit Gummi-Zement. </li><li> Legen Sie die Folie in einer angefeuchteten lichtundurchlässigen Kammer und Inkubation im Dunkeln bei 37 ° C für 24 h bis 36 h. </li></ol></li><li> Post-Hybridisierung Waschen und Fluoreszenzdetektion <ol><li> Entfernen Sie Gummizement sehr vorsichtig, ohne das Deckglas zu stören. </li><li> Legen Sie die Folie in vorgewärmten schnelle Waschlösung (0,4x SSC) bei 72 ° C (± 2 ° C) für 5 min unter konstantem Schütteln. Tauchen Abgleiten in die Waschlösung III (4x SSC / 0,1% Tween 20) und Inkubation für 1 min unter Schütteln. </li><li> Optional: Fügen Sie 80 ul Blockingreagenz(Flasche # 2 durch Hersteller mitgeliefert) auf dem Gebiet der Hybridisierung, legen Sie eine 24 mm x 60 mm Kunststoff-Deckglas, und für 30 min in einer feuchten Kammer bei 37 ° C im Dunkeln inkubiert. </li><li> Sie vorsichtig die Kunststoffdeckglas entfernen und den Schlitten mit vorgewärmten (45 ° C) Waschlösung III 5 min waschen. </li><li> Bewerben 80 ul von Cy5 Färbungsreagens (Fläschchen # 3 durch Hersteller geliefert), legen Sie eine 24 mm x 60 mm Kunststoff-Deckglas, und bei 37 ° C im Dunkeln für 40 min in einer feuchten Kammer. </li><li> Tauchen Sie den Schlitten in ein Glas Coplin Gefäß mit vorgewärmten (45 ° C) Waschlösung III (4x SSC / 0,1% Tween 20) und Inkubation bei 45 ° C in einem Wasserbad für 2 Minuten unter Schütteln. Wiederholen Sie diesen Waschschritt 3 mal. </li><li> Bewerben 80 ul von Cy5.5 Färbungsreagens (Fläschchen # 4 durch Hersteller geliefert), legen Sie eine 24 mm x 60 mm Kunststoff-Deckglas, und bei 37 ° C im Dunkeln für 40 min in einer feuchten Kammer. </li><li> Waschen Sie die Schieber 3 mal mit vorgewärmten (45 ºC) Waschlösung III. </li><li> Halten Sie die Folie in einer gekippten Position gegen ein Papiertuch, um überschüssige Flüssigkeit abfließen kann. In 20 &amp; mgr; l anti-fade DAPI-Reagenz (Fläschchen # 5 vom Hersteller geliefert) und legen Sie vorsichtig mit einem 24 mm x 60 mm Deckglas ohne Luftblasen einzuführen. Seal die Ränder des Deckglases mit Nagellack und beobachten mit einem Epifluoreszenz-Mikroskop für die Erfassung von SKY Bilder ausgestattet. </li></ol></li></ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mFISH+analysis+of+chromosomal+damage+in+bone+marrow+cells+collected+from+CBA/CaJ+mice+following+whole+body+exposure+to+heavy+ions+(56Fe+ions).">mFISH analysis of chromosomal damage in bone marrow cells collected from CBA/CaJ mice following whole body exposure to heavy ions (56Fe ions).</a> Radiat Environ Biophys. 46, 137-145 (2007).</li><li>Abrahams, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metaphase+FISHing+of+transgenic+mice+recommended:+FISH+and+SKY+define+BAC-mediated+balanced+translocation.">Metaphase FISHing of transgenic mice recommended: FISH and SKY define BAC-mediated balanced translocation.</a> Genesis. 36, 134-141 (2003).</li><li>Savage, J. R., Simpson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+scoring+of+FISH-&amp;#34;painted&amp;#34;+chromosome-type+exchange+aberrations.">On the scoring of FISH-&amp;#34;painted&amp;#34; chromosome-type exchange aberrations.</a> Mutat. Res. 307, 345-353 (1994).</li><li>Savage, J. R., Simpson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FISH+"painting"+patterns+resulting+from+complex+exchanges.">FISH "painting" patterns resulting from complex exchanges.</a> Mutat. Res. 312, 51-60 (1994).</li><li>Tucker, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PAINT:+a+proposed+nomenclature+for+structural+aberrations+detected+by+whole+chromosome+painting.">PAINT: a proposed nomenclature for structural aberrations detected by whole chromosome painting.</a> Mutat. Res. 347, 21-24 (1995).</li><li>Knehr, S., Zitzelsberger, H., Bauchinger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FISH-based+analysis+of+radiation-induced+chromosomal+aberrations+using+different+nomenclature+systems.">FISH-based analysis of radiation-induced chromosomal aberrations using different nomenclature systems.</a> Int J Radiat Biol. 73, 135-141 (1998).</li><li>Lucas, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+translocation+frequency+analysis+in+humans+decades+after+exposure+to+ionizing+radiation.">Rapid translocation frequency analysis in humans decades after exposure to ionizing radiation.</a> Int J Radiat Biol. 62, 53-63 (1992).</li><li>Braselmann, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SKY+and+FISH+analysis+of+radiation-induced+chromosome+aberrations:+a+comparison+of+whole+and+partial+genome+analysis.">SKY and FISH analysis of radiation-induced chromosome aberrations: a comparison of whole and partial genome analysis.</a> Mutat. Res. 578, 124-133 (2005).</li><li>Sokolova, I. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+a+multitarget,+multicolor+fluorescence+in+situ+hybridization+assay+for+the+detection+of+urothelial+carcinoma+in+urine.">The development of a multitarget, multicolor fluorescence in situ hybridization assay for the detection of urothelial carcinoma in urine.</a> J Mol Diagn. 2, 116-123 (2000).</li><li>Lindholm, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodosimetry+after+accidental+radiation+exposure+by+conventional+chromosome+analysis+and+FISH.">Biodosimetry after accidental radiation exposure by conventional chromosome analysis and FISH.</a> Int J Radiat Biol. 70, 647-656 (1996).</li></ol>

The authors declare that they have no competing financial interests.

Mehrere wichtige Schritte bestimmen den Erfolg von MFISH und SKY. Der erste und wichtigste Schritt ist es, die Colchicin - Behandlung für in vivo mitotischen Arrest der Knochenmark mononukleäre Zellen zu optimieren. Die Colchicin Konzentration und die Behandlungszeit der mitotischen Index sowie Chromosomenkondensation-zwei wichtige Voraussetzungen für eine effektive chromosome painting einzeln oder gemeinsam bestimmen. Eine hohe Konzentration Colchicin oder längere Behandlungszeit führt zu hoch kondensiert...

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 technique7-10. 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.

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

detection of inter-chromosomal stable aberrations by multiple fluorescence in situ hybridization (mfish) and spectral karyotyping (sky) in irradiated mice

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.

Gesamtkörperbestrahlung induziert zahlreiche Chromosomenaberrationen in den Knochenmarkzellen von bestrahlten Mäusen. Das aktuelle Protokoll wird für die in - vivo - mitotischen Arrest von Knochenmarkzellen nach Strahlenexposition optimiert, Ernte von Knochenmarkszellen von den Hinterbeinen von bestrahlten Mäusen, die Isolierung von Knochenmark mononukleären Zellen durch Dichtegradientenzentrifugation, die Vorbereitung der Metaphase Zelle ausbreitet, und die anschl...

Watch this Scientific Journal Video about Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice at JoVE

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

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.

Der Nachweis von Inter-chromosomale Stable Abberationen durch Mehrfachfluoreszenz<em&gt; In Situ</em&gt; Hybridisierung (MFISH) und Spektrale Karyotypisierung (SKY) in Bestrahlte Mäuse

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

detection-inter-chromosomal-stable-aberrations-multiple-fluorescence

Research

JoVE Journal

Genetics

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

9.8K Aufrufe.  University of Arkansas for Medical Sciences. 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. 

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

FISH for Pre-implantation Genetic Diagnosis 

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to PGD in a clinical setting.

FISH for Pre-implantation Genetic Diagnosis

This article describes the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring, applied to pre-implantation genetic diagnosis (PGD) in a clinical setting.

FISH für Präimplantations-Diagnostik

Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a ...

Forschung

Medizin

Application of Laser Micro-irradiation for Examination of Single and Double Strand Break Repair in Mammalian Cells

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular biology techniques have clarified structure, enzymatic functions, and kinetics of repair proteins, there is still a need to understand how repair is coordinated within the nucleus. Laser micro-irradiation offers a powerful tool for inducing DNA damage and monitoring the recruitment of repair proteins. Induction of DNA damage by laser micro-irradiation can occur with a range of wavelengths, and users can reliably induce single strand breaks, base lesions and double strand breaks with a range of doses. Here, laser micro-irradiation is used to examine repair of single and double strand breaks induced by two common confocal laser wavelengths, 355 nm and 405 nm. Further, proper characterization of the applied laser dose for inducing specific damage mixtures is described, so users can reproducibly perform laser micro-irradiation data acquisition and analysis.

Confocal fluorescence microscopy and laser micro-irradiation offer tools for inducing DNA damage and monitoring the response of DNA repair proteins in selected sub-nuclear areas. This technique has significantly advanced our knowledge of damage detection, signaling, and recruitment. This manuscript demonstrates these technologies to examine single and double strand break repair.

Anwendung von Mikro-Laserbestrahlung zur Prüfung von einzelnen und doppelt Strand Break Repair in Säugerzellen

Highly coordinated DNA repair pathways exist to detect, excise and replace damaged DNA bases, and coordinate repair of DNA strand breaks. While molecular ...

Krebsforschung

Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being analyzed 1, 2. This classical banding technique, however, is not reliable in identifying complex chromosomal aberrations such as those associated with cancer. To overcome the limitations of the banding technique, Spectral Karyotyping (SKY) is introduced to provide much reliable information on chromosome abnormalities.
SKY is a multicolor fluorescence in-situ hybridization (FISH) technique to detect metaphase chromosomes with spectral microscope 3, 4. SKY has been proven to be a valuable tool for the cytogenetic analysis of a broad range of chromosome abnormalities associated with a large number of genetic diseases and malignancies 5, 6. SKY involves the use of multicolor fluorescently-labelled DNA probes prepared from the degenerate oligonucleotide primers by PCR. Thus, every chromosome has a unique spectral color after in-situ hybridization with probes, which are differentially labelled with a mixture of fluorescent dyes (Rhodamine, Texas Red, Cy5, FITC and Cy5.5). The probes used for SKY consist of up to 55 chromosome specific probes 7-10.
The procedure for SKY involves several steps (Figure 1). SKY requires the availability of cells with high mitotic index from normal or diseased tissue or blood. The chromosomes of a single cell from either a freshly isolated primary cell or a cell line are spread on a glass slide. This chromosome spread is labeled with a different combination of fluorescent dyes specific for each chromosome. For probe detection and image acquisition,the spectral imaging system consists of sagnac interferometer and a CCD camera. This allows measurement of the visible light spectrum emitted from the sample and to acquire a spectral image from individual chromosomes. HiSKY, the software used to analyze the results of the captured images, provides an easy identification of chromosome anomalies. The end result is a metaphase and a karyotype classification image, in which each pair of chromosomes has a distinct color (Figure 2). This allows easy identification of chromosome identities and translocations. For more details, please visit Applied Spectral Imaging website (<a href="http://www.spectral-imaging.com/" target="_blank">http://www.spectral-imaging.com/</a>).
SKY was recently used for an identification of chromosome segregation defects and chromosome abnormalities in humans and mice with Autosomal Dominant Polycystic Kidney Disease (ADPKD), a genetic disease characterized by dysfunction in primary cilia 11-13. Using this technique, we demonstrated the presence of abnormal chromosome segregation and chromosomal defects in ADPKD patients and mouse models 14. Further analyses using SKY not only allowed us to identify chromosomal number and identity, but also to accurately detect very complex chromosomal aberrations such as chromosome deletions and translocations (Figure 2).

Spectral Karyotyping (SKY) is an advanced cytogenetics technique to identify genomic and chromosomal aberrations. This technique takes advantage of chromosome painting probes, which allow classification of all chromosomes. SKY can also identify complex chromosome aberrations and segregation defects in mice and humans with various diseases, including polycystic kidney disease.

Spektrale Karyotypisierung zu Chromosomenanomalien in Menschen und Mäusen mit Kidney Disease Study

Conventional method to identify and classify individual chromosomes depends on the unique banding pattern of each chromosome in a specific species being ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

Live Cell Imaging der Segregation der Chromosomen während der Mitose

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Entwicklungsbiologie

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Schnelle Analyse von Chromosomenaberrationen in Maus-B-Lymphozyten durch PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Immunologie und Infektion

Identifizierung von DNA-Schäden in prophase-arrestierten Eizellen von Mäusen

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis. This protracted state of arrest makes them extremely susceptible to accumulating DNA-damaging insults, which affect the genetic integrity of the female gametes and, therefore, the genetic integrity of the future embryo. 
Consequently, the development of an accurate method to detect DNA damage, which is the first step for the establishment of DNA damage response mechanisms, is of vital importance. This paper describes a common protocol to test the presence and progress of DNA damage in prophase-arrested oocytes during a period of 20 h. Specifically, we dissect mouse ovaries, retrieve the cumulus-oocyte complexes (COCs), remove the cumulus cells from the COCs, and culture the oocytes in &#924;2 medium containing 3-isobutyl-1-methylxanthine to maintain the state of arrest. Thereafter, the oocytes are treated with the cytotoxic, antineoplasmic drug, etoposide, to engender double-strand breaks (DSBs). 
By using immunofluorescence and confocal microscopy, we detect and quantify the levels of the core protein &#947;H2AX, which is the phosphorylated form of the histone H2AX. H2AX becomes phosphorylated at the sites of DSBs after DNA damage. The inability to restore DNA integrity following DNA damage in oocytes can lead to infertility, birth defects, and increased rates of spontaneous abortions. Therefore, the understanding of DNA damage response mechanisms and, at the same time, the establishment of an intact method for studying these mechanisms are essential for reproductive biology research.

Autor im Rampenlicht: Verständnis der DNA-Schadensreaktion in Säugetier-Eizellen und Präimplantationsembryonen 

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

Nachweis von DNA-Doppelstrangbrüchen in Maus-Eizellen

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain ...

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

Chromosomics: Erkennung von numerischen und strukturellen Veränderungen in alle 24 menschlichen Chromosomen gleichzeitig unter Verwendung eines neuartigen OctoChrome FISH-Test

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

Biologie

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Chromosome Replizieren Timing mit Fluorescent Kombiniert<em&gt; In situ</em&gt; Hybridisierung

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Der Nachweis von Inter-chromosomale Stable Abberationen durch Mehrfachfluoreszenz

Zusammenfassung

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