Spectral karyotyping analysis of human and mouse chromosomes
Spectral karyotyping analysis of human and mouse chromosomesHesed M Padilla Nash1,Top of page
Cytogenetic studies in cancer are complicated by the fact that many chromosomal aberrations are often difficult to characterize. Initially, researchers relied on classical banding (G , Q and C banding) methods1, 2, 3. These methods helped distinguish individual chromosomes by visualizing physical landmarks (called bands) and provided chromosome specific banding patterns for each species examined. Cytogenetic techniques became much more versatile through the introduction of hybridization of fluorescently labeled region specific DNA probes. This molecular cytogenetic technique is now widely known as FISH4,
card for humanity, 5, 6.
SKY is also based on the hybridization of fluorescently labeled DNA probes; however, the probe used is complex, generally consisting of up to 55 individually generated chromosome specific probes. SKY has proven exceedingly valuable for the comprehensive analysis of cytogenetic abnormalities associated with malignant disease and has been applied to a large series of samples derived from hematological malignancies and solid tumors7, 8, 9, 10, 11. In fact, since its invention in 1996, more than 500 papers have been published that applied SKY for the analysis of various chromosomal preparations. Alternatively,
cards against humanity best expansion, M FISH13 can be used for characterizing karyotypes14. Although the hybridization principle is identical to SKY, M FISH uses a set of fluorochrome specific optic filters (five or seven different filters), rather than the single custom designed filter used in SKY. Both SKY and M FISH achieve the same goal is, identification of individual chromosomes in different colors. Both methods are more costly than conventional banding methods, as they require expensive materials (probes), equipment and software.
The systematic analysis of chromosomal abnormalities in cancer cells using SKY allows for the characterization of novel and hidden chromosomal translocations, identification of complex rearrangements15 and reconstruction of clonal evolution events during cancer progression, and it has revealed the role of unstable chromosome rearrangements, such as jumping translocations, occurring as tissue specific genomic imbalances11. Although SKY allows for the identification of which particular piece of DNA may be contained within a chromosome aberration, the spectral image alone does not provide information as to the specific region of the chromosome localized within a rearrangement. For this, one must rely on the banding pattern of 4,6 diamidino 2 phenylindole (DAPI). Good quality chromosome preparations are also key to the success of SKY analysis, if the chromosome preparations are of inferior quality example, if the slides are old or have too much cytoplasm surrounding the chromosomes analysis can be less than optimal. Thus, the strengths and limitations of SKY analysis are somewhat dependent on the skills of the user. Maximizing the amount of information gained through the use of this technique is greatly facilitated by a general knowledge and expertise in cytogenetics is, familiarity with the chromosomal banding patterns of the species being analyzed, as well as an understanding of the principles of how the software interprets the acquired information. For assistance in both identification of the banding patterns and determination of breakpoints of aberrant human chromosomes, researchers use the nomenclature rules defined in ref. 16.
SKY greatly assists in identifying chromosomal regions involved in homogenous staining regions and double minute chromosomes, regardless of size and numbers. However, with respect to homogenous staining regions and double minute chromosomes, again, SKY has its limitations, in that these aberrations often contain multiple genes and/or DNA regions that are tightly linked; resolving these details therefore often requires additional FISH hybridizations with either gene loci probes or specific chromosome or chromosome arm paints17. SKY has also proven to be very useful for the study of constitutional chromosome abnormalities arising from de novo balanced translocations (in which chromosomal regions are relocated in a reciprocal manner and no genetic material is lost) and unbalanced translocations (which are, by definition, associated with genomic imbalances) that occasionally involve chromosome exchanges between very small DNA regions, especially at the telomeres18, 19, 20. In prenatal diagnosis, the difference between the outcome of a balanced translocation and an unbalanced one may represent the difference between a normal child and one with mild to severe birth defects. SKY is useful in determining the origin of supernumerary marker chromosomes that are occasionally seen in prenatal specimens (appearing as unbanded small chromosome fragments using conventional banding methods). It is imperative that the chromosomal origin of these markers be determined, as genomic imbalances may result in birth defects and or mental retardation21, and some markers may have considerable prognostic significance.
SKY has also been applied for the reconstruction of chromosomal rearrangements that have occurred during the course of chromosome evolution, a field referred to as comparative cytogenetics22. By using SKY, one can determine and compare homologous regions of genomes between closely related species example, between human and a gibbon (Hylobates concolor)23. These analyses can be complemented by performing the reverse experiment is, hybridizing differentially labeled flow sorted gibbon chromosomes onto human metaphase spreads. This is referred to as cross species hybridization24. Recently, SKY analysis has also been used in other species, such as rat. Newly developed rat probes have been used to determine the homologous regions of rat, mouse and human chromosomes, as well as to characterize the karyotypes of rat tumors25.
The study of chromosomal aberrations in mice is extremely demanding, because the chromosomes of this species are all acrocentric (that is, the primary constriction, or centromere, is located at one end of the chromosome) and of similar size and morphology. Therefore, subtle aberrations such as insertions or translocations involving small regions are often unrecognizable. Murine models of human carcinogenesis are widely used to delineate genetic mechanisms that determine tumor initiation and progression26. SKY as a genome scanning method for detection of chromosomal aberrations has now been applied to a wide variety of murine models of human cancer, including numerous models for hematological malignancies and solid tumors27, 28, 29, 30. Although SKY has been applied to vertebrate organisms closely related to human or mouse, thus far commercially made SKY probes and analysis software are only available for human, mouse and rat.
Experimental designThe protocol we present uses SkyPaint probes (Applied Spectral Imaging). The SKY analysis protocol described herein consists of six parts: (i) preparation of metaphase chromosome spreads ( 1 (ii) slide pretreatment and slide and probe denaturation (Box 1 and 21 (iii) hybridization ( 27 and (iv) detection ( 30 of the SKY probes; and (v) tips for image acquisition and (vi) image analysis (Step 50) (see Fig. 1). Preparation of metaphase chromosome spreads for SKY analysis requires 1 d. Slide pretreatment and denaturation of both the slide and the probe takes 2 h, at which point one can pause before continuing with the hybridization steps. The hybridization protocol, which includes denaturation of the SKY probe as well as that of the target metaphase chromosomes on the slide, is performed on day 1. For the actual hybridization of human or mouse SKY probes, the procedure continues for 48 h at 37 The subsequent detection steps take approximately 5 h (see Fig. 1). Image acquisition and analysis can take place immediately after the detection steps, but can also be delayed and the slides can be stored in the dark at 4 We highly recommend analyzing the slides within 1 week of completion of the SKY detection procedures.
Figure 1: Protocol timeline flowchart.
Shown are the steps in a SKY hybridization and analysis.
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Preparation of metaphase spreads for SKY. This requires (i) knowledge of the ideal culture conditions of the specimen studied to obtain actively dividing cells, and (ii) recognizing the conditions necessary to yield spreads free of cytoplasm, which include (iii) the optimum humidity required for the individual chromosome preparations to ensure proper spreading of chromosomes onto glass slides and eliminate the possibility of chromosomes overlapping31. If none of these conditions are met, hybridization with SKY and or FISH probes will be suboptimal, thereby impeding an accurate analysis of the results. Excess cytoplasm surrounding the chromosomes can be reduced by pretreating the slides containing the metaphase preparations with enzymes such as trypsin or pepsin that digest the surrounding cellular proteins. This facilitates access of the DNA probe to its target sequence on the chromosome as well as reducing the amount of background signal that arises when the DNA probe nonspecifically binds the proteins (see Fig. 2). However, overtreatment of the slides can also reduce hybridization efficiency through degradation of the target DNA or excessive removal of chromosome associated histone proteins, leading to a distortion of the chromosome morphology. Should this occur, we recommend repeating this phase of the experiment with new slides.
Figure 2: Effects of humidity and removal of excess cytoplasm by slide pretreatment on hybridization efficiency.
(a) When cells are not kept in the hypotonic solution for enough time, the chromosomes become trapped inside the cytoplasm (white arrows). Altering the humidity at which the metaphase preparation is dropped onto the microscope slide may sometimes alleviate this problem; otherwise, hybridization efficiency will be compromised. (b) When normal human metaphase spreads are pretreated with pepsin and hybridized with a whole chromosome paint probe labeled with tetramethyl rhodamine isothyocyanate (TRITC), the hybridization will be successful. (c) Here,
against humanity, a whole chromosome paint labeled with TRITC was hybridized to a different normal human metaphase spread and the slide was not pretreated with pepsin. Note the weaker intensity of the hybridization signal (red color).
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SKY probe generation and labeling. SKY probes consist of a cocktail of chromosome specific painting probes of the target species. The chromosome painting probes used for SKY analysis of human or mouse genomes are prepared by amplification of flow sorted chromosomes using degenerate oligonucleotide (DOP) PCR32. ASI produced SkyPaint probes undergo stringent quality controls and their chromosome specificity is tested by hybridization of all probes individually using FISH. Probes are labeled with nucleotide analogs,
cards against humanity sale, either conjugated directly to fluorochromes, such as Spectrum orange, Rhodamine green or Texas Red, or indirectly via small nonfluorescent molecules example, biotin or digoxygenin, which are visualized using avidin and antibody conjugated fluorochromes. As five distinct fluorochromes are distinguishable from one another using the SKY system, each chromosome is labeled with a unique combination of these tagged nucleotides. To further enhance the chromosome specificity of the probe cocktail, the labeled chromosome painting probes are denatured and combined with an excess of CotI DNA (which represents a fraction of the genome enriched for highly repetitive elements, such as long and short interspersed elements, Alu repeats and so forth, that are distributed throughout all the chromosomes). This step is known as preannealing and ensures that any labeled non repetitive elements in the chromosome painting probe form double stranded DNA with their complementary sequences found in the CotI DNA. These repetetive elements are thereby prevented from hybridizing to the metaphase chromosome spreads.
Probe hybridization and detection for SKY analysis. The principles of SKY probe hybridization and detection are very similar to those of FISH. The probe is placed in direct contact with denatured metaphase chromosomes (which are attached to glass slides), allowing for Watson Crick base pairing to occur between complementary sequences in the labeled probe and the metaphase chromosomes. This hybridization step is allowed to proceed for 24 h to 3 d, depending on the age and conditions of the starting material on the slide (older slides tend to require longer hybridization times). The slides are then washed to remove unhybridized probe and nonspecifically hybridized probe (that is, probe that has formed double bonds with sequences in the chromosomes that are not fully complementary). Those chromosomes labeled directly with fluorescently conjugated nucleotides do not require anything further for their visualization under the microscope. Nonfluorescensly labeled probes, in contrast, require further detection with a molecule such as Avidin (for the detection of biotin) or an antibody, either of which has a fluorochrome attached to it. In addition to the chromosome specific fluorescent label, we use DAPI to introduce a grayscale chromosome banding pattern that is similar to Giemsa trypsin (G) banding.
Image acquisition and analysis. This is accomplished by viewing the slides with an epifluorescence microscope equipped with hardware and software designed for visualizing SKY images (see Fig. 3 and ref. 36). The epifluorescence microscope should be equipped with a oil immersion objective, a single custom designed triple band pass filter (SKY filter cube) that allows for the simultaneous excitation of all fluorochromes, and a DAPI filter cube. Image acquisition requires illumination from a 150 W xenon lamp. The emitted light is passed through the collection optics and then the Sagnac interferometer, where an optical path difference is created (see Fig. 3). The interferogram measures every pixel detected by the CCD camera. Using Fourier transformation, it mathematically retrieves the wavelengths of the emitted light from the combinatorially labeled chromosomes and thereby identifies a spectral signature for each pixel of the CCD camera image.