1Department of Pharmacology, University of Toledo, College of Pharmacy and Pharmaceutical Sciences, 2Department of Emergency and Intensive Care, ProMedica Sponsored Research
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AbouAlaiwi, W. A., Rodriguez, I., Nauli, S. M. Spectral Karyotyping to Study Chromosome Abnormalities in Humans and Mice with Polycystic Kidney Disease. J. Vis. Exp. (60), e3887, doi:10.3791/3887 (2012).
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).
1. Cell pretreatment and metaphase preparation
2. Slide pretreatment (pepsin treatment)
3. Chromosome and probe denaturation and hybridization
4. Fluorescent probe detection
5. Image acquisition and analysis
6. Representative Results
A complete SKY procedure usually takes about one week time (Figure 1). This includes image acquisition and analysis provided that the metaphase cells are in adequate supply. Karyotyping analysis reveals normal mouse karyotype (40,XY) of cells from wild type mice (Figure 2a). In contrast, cells from Pkd1-/- mouse (PKD mouse model) shows a significant increase in chromosome number and structural abnormalities, such as chromosome deletions (Chromosome # 8) and translocations (chromosomes # 11 and 19) (Figure 2b). We also analyzed vascular tissues from the ADPKD patients. A simple study by counting the chromosomal numbers indicated that non-ADPKD and some ADPKD vascular samples had normal chromosomal numbers of 23 pairs (Figure 2c). In general, however, we observed failure of chromosomal segregation, resulting in 46 pairs of chromosomes in ADPKD samples instead of 23 pairs (Figure 2d).
Figure 1. SKY protocol flowchart. Flow chart of the SKY protocol illustrates steps to complete an experiment starting from cell pretreatment and metaphase preparations to image acquisition and analysis. An approximate one-week timeline is presented on the left with step-by-step procedures for each day.
Figure 2. Spectral karyotyping on immortalized mouse cell lines and freshly isolated human primary cells. Color and inverted DAPI images of individual chromosomes are shown before the chromosomal sorting. After sorting, chromosomes are presented in the "classification" table. a) SKY image of a metaphase spread from wild type mouse contains normal karyotype (40, XY). b) SKY image of a metaphase spread from Pkd1-/- mouse shows abnormal chromosome number (68 instead of 40) and chromosome abnormalities such as chromosome deletions (chromosome #8) and chromosome translocations (chromosomes #11 and 19). c) SKY image of a metaphase spread from the vascular tissue of a non-ADPKD patient has normal karyotype (46, XX). d) SKY image of a metaphase spread from the vascular tissue of an ADPKD patient contains abnormal karyotype (92, XXXX). Parts of the data have been previously reported and reused with permission 14.
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 cytogenetics laboratories, including ours.
Among the key steps in the protocol, a good quality metaphase preparation is extremely important to have a successful SKY analysis. For example, if the chromosomes have too much cytoplasm or if the slides are too old, the quality of the images could be compromised. As seen in our movie clip, the content of cytoplasm can be easily identified using a high resolution of differential interference contrast microscopy technique. In addition, the availability of actively dividing cells is a prerequisite for SKY analysis; this can be overcome by treatment with colcemid for a longer time or an addition of other growth factors to the cells. The overall success rate of SKY is largely dependent on the skills and experience of the user.
SKY offers a powerful and reliable tool for chromosomal analysis, including structural changes in a single chromosome. In addition to cellular polyploidy, SKY offers a possibility to study chromosome insertions, deletions, duplications and translocations. For example, SKY allows identification of novel and hidden chromosomal aberrations and identification of complex rearrangements during cancer development and progression 15. Furthermore, SKY has been applied to a research field within comparative cytogenetics, which studies chromosomal rearrangements during evolution 4. Using SKY technique, our laboratory becomes the first to identify chromosome abnormalities in mice and humans with polycystic kidney disease (Figure 2). There is no doubt that SKY will be beneficial in many other chromosomal associated diseases. In fact, we propose that SKY would also be useful in many cilia-related pathogenesis, where cilia have been shown to regulate cell division 14.
Because mouse and rat are important animal models to study the mechanism of a disease and to perform a functional assessment in vivo, the development of mouse and rat probes for SKY analysis has allowed routine karyotyping of mouse and rat chromosomes. At present, SKY is therefore limited to chromosomal studies in human, mouse and rat. However, as more research laboratories use other species to study genomic compositions, it is foreseeable that more chromosome painting probes for different organisms will soon be commercially available.
No conflicts of interest declared.
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 & ProMedica Translational Research Stimulation Award to Dr. Surya Nauli.
|Fetal bovine serum (FBS)||Hyclone||SH30088-03|
|Penecillin/Streptomycin||Thermo Fisher Scientific, Inc.||SV30010|
|Colcemid||Roche Group||10 295 892 001||10 μg/ml|
|Phosphate buffered saline||Thermo Fisher Scientific, Inc.||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 Fisher Scientific, Inc.||SH30236.01|
|Acetic acid||Fisher Scientific||A38-212|
|RNase A||Roche Group||10 109 169 001|
|37% Formaldehyde||Mallinckrodt Baker Inc.||2106-02|
|20X SSC||Promega Corp.||V4261|
|Formamide||Fluka||47671||prepare just before use|
|Microscope glass slides||Fisher Scientific||12-549|
|Microscope cover glass 24x60mm||VWR international||16004-312|
|Hybridization/ humidifiedchamber/Tray||Simport||M920-2||put wet paper towels at the bottom|
|Shaking platform/Orbital shaker||Bellco Glass|
|Shaking/water bath||Precision Scientific|
|DAPI filter cube||Chroma Technology Corp.|
|SKY filter cube||Chroma Technology Corp.|
|SpectraCube||Applied Spectral Imaging|
|Inverted cell culture microscope||Nikon Instruments||Nikon Eclipse TS100|
|Fluorescence microscope||Olympus Corporation||IX70||60X oil|