Authors would like to thank Brian Muntean, Shao Lo, Maki Takahashi and Blair Mell for their technical assistance. This work was supported by awards from the NIH (DK080640) and The University of Toledo &amp; ProMedica Translational Research Stimulation Award to Dr. Surya Nauli.

1. Cell pretreatment and metaphase preparation
<ol>
	<li>The cells are grown in Dulbecco's Modification of Eagle's Medium (DMEM) containing 10-15% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37&deg;C with 5% CO2 incubator, until they reach 70-80% confluency.</li>
	<li>Treat the cells with colcemid solution at 0.05 &mu;g/ml for 30-60 min.</li>
	<li>Collect the medium containing any floating cells in 50-ml sterile falcon centrifuge tubes. Rinse the cells on the plate with sterile1X-PBS. After in...

<ol>
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R., Matsui, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+disruption+of+cytosine+DNA+methyltransferase+enzymes+induces+chromosomal+instability+in+human+cancer+cells.">Genetic disruption of cytosine DNA methyltransferase enzymes induces chromosomal instability in human cancer cells.</a> Cancer Research. 65, 8635-8639 (2005).</li><li>Matsui, S., Faitar, S. L., Rossi, M. R., Cowell, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+spectral+karyotyping+to+the+analysis+of+the+human+chromosome+complement+of+interspecies+somatic+cell+hybrids.">Application of spectral karyotyping to the analysis of the human chromosome complement of interspecies somatic cell hybrids.</a> Cancer Genetics and Cytogenetics. 142, 30-35 (2003).</li><li>Nestor, A. L., Hollopeter, S. L., Matsui, S. I., Allison, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+genetic+complementation+controlling+the+chromosomal+abnormalities+and+loss+of+heterozygosity+formation+in+cancer.">A model for genetic complementation controlling the chromosomal abnormalities and loss of heterozygosity formation in cancer.</a> Cytogenetic and Genome Research. 116, 235-247 (2007).</li><li>Ried, T., Liyanage, M., du Manoir, S., Heselmeyer, K., Auer, K., Macville, M., Schrock, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cytogenetics+revisited:+comparative+genomic+hybridization+and+spectral+karyotyping.">Tumor cytogenetics revisited: comparative genomic hybridization and spectral karyotyping.</a> Journal of Molecular Medicine (Berlin, Germany). 75, 801-814 (1997).</li><li>AbouAlaiwi, W. A., Takahashi, M., Mell, B. R., Jones, T. J., Ratnam, S., Kolb, R. J., Nauli, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ciliary+polycystin-2+is+a+mechanosensitive+calcium+channel+involved+in+nitric+oxide+signaling+cascades.">Ciliary polycystin-2 is a mechanosensitive calcium channel involved in nitric oxide signaling cascades.</a> Circulation Research. 104, 860-869 (2009).</li><li>Nauli, S. M., Alenghat, F. J., Luo, Y., Williams, E., Vassilev, P., Li, X., Elia, A. E., Lu, W., Brown, E. M., Quinn, S. J., Ingber, D. E., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycystins+1+and+2+mediate+mechanosensation+in+the+primary+cilium+of+kidney+cells.">Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells.</a> Nature Genetics. 33, 129-137 (2003).</li><li>Nauli, S. M., Kawanabe, Y., Kaminski, J. J., Pearce, W. J., Ingber, D. E., Zhou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cilia+are+fluid+shear+sensors+that+regulate+calcium+signaling+and+nitric+oxide+production+through+polycystin-1.">Endothelial cilia are fluid shear sensors that regulate calcium signaling and nitric oxide production through polycystin-1.</a> Circulation. 117, 1161-1171 (2008).</li><li>AbouAlaiwi, W. A., Ratnam, S., Booth, R. L., Shah, J. V., Nauli, S. 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Spectral Karyotyping (SKY) is a cytogenetics technique used in studying genomic and chromosomal compositions. This technique takes advantage of chromosome painting probes, and the detection of these probes are acquired through a sagnac interferometer. The complete SKY process usually takes about one week, and it involves several key steps (Figure 1). The SKY uses a standard protocol, which was first described by Padilla-Nash et al 3. The protocol has since been adapted by various cyto...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Cellgro Mediatech Inc.</td>
			<td>10-013-CV</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30088-03</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penecillin/Streptomycin</td>
			<td>Thermo Scientific</td>
			<td>SV30010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Colcemid</td>
			<td>Roche Diagnostics</td>
			<td>10 295 892 001</td>
			<td>10 &mu;g/ml</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Fisher Scientific</td>
			<td>A144-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific</td>
			<td>S77375-1</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Thermo Scientific</td>
			<td>SH30256-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SKY paint probe kit (Human)</td>
			<td>Applied Spectral Imaging</td>
			<td>SKY000028</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SKY paint probe kit (Mouse)</td>
			<td>Applied Spectral Imaging</td>
			<td>SKY000030</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Concentrated antibody detection kit</td>
			<td>Applied Spectral Imaging</td>
			<td>SKY000033</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Thermo Scientific</td>
			<td>SH30236.01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A433P-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Fisher Scientific</td>
			<td>A38-212</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RNase A</td>
			<td>Roche Diagnostics</td>
			<td>10 109 169 001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pepsin</td>
			<td>Sigma-Aldrich</td>
			<td>P6887-5G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Fluka</td>
			<td>63069-500ML</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>37% Formaldehyde</td>
			<td>Mallinckrodt Baker</td>
			<td>2106-02</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>20X SSC</td>
			<td>Promega</td>
			<td>V4261</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Fluka</td>
			<td>47671</td>
			<td>prepare just before use</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope glass slides</td>
			<td>Fisher Scientific</td>
			<td>12-549</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Microscope cover glass 24x60mm</td>
			<td>VWR Vista Vision</td>
			<td>16004-312</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rubber cement</td>
			<td>Elmer's (local retail store)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Hybridization/ humidifiedchamber/Tray</td>
			<td>Simport</td>
			<td>M920-2</td>
			<td>put wet paper towels at the bottom</td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>Eppendorf</td>
			<td>Epgradient S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Shaking platform/Orbital shaker</td>
			<td>Bellco Biotechnology</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Shaking/water bath</td>
			<td>Precision Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DAPI filter cube</td>
			<td>Chroma Technology</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SKY filter cube</td>
			<td>Chroma Technology</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SpectraCube</td>
			<td>Applied Spectral Imaging</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Inverted cell culture microscope</td>
			<td>Nikon</td>
			<td>Nikon Eclipse TS100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Olympus</td>
			<td>IX70</td>
			<td>60X oil</td>
		</tr>
	</tbody>
</table>

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).

Watch this Scientific Journal Video about Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease at JoVE.com

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

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.

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

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Research

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Medicine

38.0K visualizações.  University of Toledo, College of Pharmacy and Pharmaceutical Sciences. Cariotipagem espectral (SKY) é uma técnica avançada citogenética para identificar as aberrações cromossômicas e cromossômicas. Essa técnica leva vantagem das sondas cromossômicas de pintura, que permitem a classificação de todos os cromossomos. SKY também pode identificar aberrações cromossômicas e defeitos complexos de segregação em camundongos e humanos, com várias doenças, incluindo doença renal policística.