Спектральный Кариотипирование по изучению хромосомных аномалий у человека и мыши с поликистозом почек
Summary
Спектральный Кариотипирование (SKY) является передовой техникой цитогенетики для идентификации геномных и хромосомных аберраций. Эта техника использует датчики хромосомы картины, которые позволяют классификации всех хромосом. SKY можно также определить комплекс хромосомных аберраций и разделение дефектов у мышей и людей с различными заболеваниями, в том числе поликистоз почек.
Abstract
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 (http://www.spectral-imaging.com/).
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).
Protocol
Discussion
Спектральный Кариотипирование (SKY) является цитогенетики техника, используемая в изучении генома и хромосомные композиций. Эта техника использует датчики хромосомы живописи, и обнаружение этих зондов, полученных в результате Саньяка интерферометра. Весь процесс SKY обычно занимает ок?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Авторы хотели бы поблагодарить Брайана Мунтян, Шао вот, Маки Такахаши и Блэр Мелл за техническую помощь. Эта работа была поддержана награды от NIH (DK080640) и Университет Толедо и ProMedica Поступательное премии стимулирование исследований доктора Сурья Наули.
Materials
| Name of the reagent | Company | Catalog number | Comments |
| DMEM | Cellgro Mediatech Inc. | 10-013-CV | |
| Fetal bovine serum (FBS) | Hyclone | SH30088-03 | |
| Penecillin/Streptomycin | Thermo Scientific | SV30010 | |
| Colcemid | Roche Diagnostics | 10 295 892 001 | 10 μg/ml |
| HCl | Fisher Scientific | A144-500 | |
| KCl | Fisher Scientific | S77375-1 | |
| Phosphate buffered saline | Thermo Scientific | SH30256-01 | |
| SKY paint probe kit (Human) | Applied Spectral Imaging | SKY000028 | |
| SKY paint probe kit (Mouse) | Applied Spectral Imaging | SKY000030 | |
| Concentrated antibody detection kit | Applied Spectral Imaging | SKY000033 | |
| Trypsin | Thermo Scientific | SH30236.01 | |
| Methanol | Fisher Scientific | A433P-4 | |
| Acetic acid | Fisher Scientific | A38-212 | |
| RNase A | Roche Diagnostics | 10 109 169 001 | |
| Pepsin | Sigma-Aldrich | P6887-5G | |
| MgCl2 | Fluka | 63069-500ML | |
| 37% Formaldehyde | Mallinckrodt Baker | 2106-02 | |
| 20X SSC | Promega | V4261 | |
| Formamide | Fluka | 47671 | prepare just before use |
| Tween-20 | Fisher Scientific | BP337-500 | |
| Microscope glass slides | Fisher Scientific | 12-549 | |
| Microscope cover glass 24x60mm | VWR Vista Vision | 16004-312 | |
| Rubber cement | Elmer’s (local retail store) | ||
| Hybridization/ humidifiedchamber/Tray | Simport | M920-2 | put wet paper towels at the bottom |
| Thermocycler | Eppendorf | Epgradient S | |
| Shaking platform/Orbital shaker | Bellco Biotechnology | ||
| Shaking/water bath | Precision Scientific | ||
| DAPI filter cube | Chroma Technology | ||
| SKY filter cube | Chroma Technology | ||
| SpectraCube | Applied Spectral Imaging | ||
| Inverted cell culture microscope | Nikon | Nikon Eclipse TS100 | |
| Fluorescence microscope | Olympus | IX70 | 60X oil |
Riferimenti
- Caspersson, T., Farber, S., Foley, G. E., Kudynowski, J., Modest, E. J., Simonsson, E., Wagh, U., Zech, L. Chemical differentiation along metaphase chromosomes. Experimental Cell Research. 49, 219-222 (1968).
- Seabright, M. A rapid banding technique for human chromosomes. Lancet. 2, 971-972 (1971).
- Padilla-Nash, H. M., Barenboim-Stapleton, L., Difilippantonio, M. J., Ried, T. Spectral karyotyping analysis of human and mouse chromosomes. Nature Protocols. 1, 3129-3142 (2006).
- Schrock, E., Zschieschang, P., O’Brien, P., Helmrich, A., Hardt, T., Matthaei, A., Stout-Weider, K. Spectral karyotyping of human, mouse, rat and ape chromosomes–applications for genetic diagnostics and research. Cytogenetic and Genome Research. 114, 199-221 (2006).
- Padilla-Nash, H. M., Heselmeyer-Haddad, K., Wangsa, D., Zhang, H., Ghadimi, B. M., Macville, M., Augustus, M., Schrock, E., Hilgenfeld, E., Ried, T. Jumping translocations are common in solid tumor cell lines and result in recurrent fusions of whole chromosome arms. Genes, Chromosomes & Cancer. 30, 349-363 (2001).
- Veldman, T., Vignon, C., Schrock, E., Rowley, J. D., Ried, T. Hidden chromosome abnormalities in haematological malignancies detected by multicolour spectral karyotyping. Nature Genetics. 15, 406-410 (1997).
- Karpf, A. R., Matsui, S. Genetic disruption of cytosine DNA methyltransferase enzymes induces chromosomal instability in human cancer cells. Ricerca sul cancro. 65, 8635-8639 (2005).
- Matsui, S., Faitar, S. L., Rossi, M. R., Cowell, J. K. Application of spectral karyotyping to the analysis of the human chromosome complement of interspecies somatic cell hybrids. Cancer Genetics and Cytogenetics. 142, 30-35 (2003).
- Nestor, A. L., Hollopeter, S. L., Matsui, S. I., Allison, D. A model for genetic complementation controlling the chromosomal abnormalities and loss of heterozygosity formation in cancer. Cytogenetic and Genome Research. 116, 235-247 (2007).
- Ried, T., Liyanage, M., du Manoir, S., Heselmeyer, K., Auer, K., Macville, M., Schrock, E. Tumor cytogenetics revisited: comparative genomic hybridization and spectral karyotyping. Journal of Molecular Medicine (Berlin, Germany). 75, 801-814 (1997).
- AbouAlaiwi, W. A., Takahashi, M., Mell, B. R., Jones, T. J., Ratnam, S., Kolb, R. J., Nauli, S. M. Ciliary polycystin-2 is a mechanosensitive calcium channel involved in nitric oxide signaling cascades. Circulation Research. 104, 860-869 (2009).
- 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. Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells. Nature Genetics. 33, 129-137 (2003).
- Nauli, S. M., Kawanabe, Y., Kaminski, J. J., Pearce, W. J., Ingber, D. E., Zhou, J. Endothelial cilia are fluid shear sensors that regulate calcium signaling and nitric oxide production through polycystin-1. Circulation. 117, 1161-1171 (2008).
- AbouAlaiwi, W. A., Ratnam, S., Booth, R. L., Shah, J. V., Nauli, S. M. Endothelial cells from humans and mice with polycystic kidney disease are characterized by polyploidy and chromosome segregation defects through survivin down-regulation. Human Molecular Genetics. 20, 354-367 (2011).
- Padilla-Nash, H. M., Nash, W. G., Padilla, G. M., Roberson, K. M., Robertson, C. N., Macville, M., Schrock, E., Ried, T. Molecular cytogenetic analysis of the bladder carcinoma cell line BK-10 by spectral karyotyping. Genes, Chromosomes & Cancer. 25, 53-59 (1999).
