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°C with 5% CO2 incubator, until they reach 70-80% confluency.</li>
	<li>Treat the cells with colcemid solution at 0.05 μg/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 incubating with sterile trypsin for 1-2 min, harvest and collect the remaining cells into the same tube.</li>
	<li>Spin the tubes at 1000 r.p.m. for 5 min, aspirate the supernatant leaving 0.5 ml and loosen pellet by flicking with finger only.</li>
	<li>Depending on the pellet size, add 5-10 ml of hypotonic solution of 0.56% KCl in dH2O and incubate the suspension at 37°C for 30-45 min.</li>
	<li>Add one drop of methanol/acetic acid (3:1 vol/vol) per ml of hypotonic cell suspension and invert the tube gently for mixing.</li>
	<li>Centrifuge at 1200 r.p.m. for 5 min and collect pellet as step 1.4, then add 5 ml of fresh methanol/acetic acid (3:1 vol/vol) fixative solution dropwise while flicking the pellet continously. This procedure is critical so that the metaphase spreads will not be trapped in the cells' clumps which would compromise the experiment.</li>
	<li>Centrifuge again at 1200 r.p.m. for 5 min and add 5-10 ml of ice-cold methanol/acetic acid fixative along the wall of the tube. At this stage, if needed, the cells can be stored in fixative solution in a tightened and sealed tube at -20°C for short term or -80°C for long term (years) for future use.</li>
	<li>Clean slides in absolute ethanol, then dip the slide in dH2O for approximately10X in order to form a sheath of water on the surface of the slide. Place the slide on a glass plate and drop 15-20μl of cell suspension (from step 1.8) from 10" above the slide. Place the slide in a water bath set at 65-70°C for 1-2 min and allow to dry.</li>
	<li>Check the slide under a light microscope using 10X and 40X dry objectives making sure there is metaphase chromosomes and the spreads are evenly spaced. Check for cytoplasm presence surrounding the chromosomes. If cytoplasm is present proceed with slide pretreatment (pepsin digestion), if no cytoplasm is present and the chromosomes have good morphology, then there is no need for slide pretreatment.</li>
</ol>
2. Slide pretreatment (pepsin treatment)
<ol>
	<li>Apply 120 μl of 1:200 RNase solution (20 mg/ml) dissolved in 2X-SSC onto a 24 mm x 60 mm microscope coverglass and invert the metaphase slide face down on the coverglass then gently invert the metaphase slide face up and incubate at 37°C for 45 min.</li>
	<li>Carefully remove the coverglass without scratching the slide and wash in 2X-SSC buffer in a coplin jar every 5 min for 15 min with shaking.</li>
	<li>Add 5-15 μl of pepsin stock solution (100 mg/ml in dH2O) into a clean beaker and then add 100 ml of prewarmed (37°C) 0.01M HCl. It is important that the pepsin is added into a clean beaker first and not directly into the HCl solution, otherwise it wouldn't dissolve in solution. Incubate the slide in a coplin jar containing the HCl/pepsin solution at 37°C for 3-5 min. This step is very critical as too much digestion will cause the chromosomes to be overdigested and too little digestion will leave the cytoplasm undigested which might lead to non-specific binding of the probe and interfere with the hybridization signal.</li>
	<li>Wash the slide in a coplin jar containing 100 ml 1X-PBS for 5 min twice at room temperature.</li>
	<li>Wash the slide in a coplin jar containing 100 ml 1X-PBS/MgCl2 for 5 min at room temperature (50 ml of 1M MgCl2 in 950 ml of 1X-PBS).</li>
	<li>Place the slide in a coplin jar containing 100 ml 1% formaldehyde for 10 min at room temperature (1.7 ml of 37% formaldehyde in 100 ml of 1X-PBS/MgCl2).</li>
	<li>Wash the slide in a coplin jar containing 100 ml 1X-PBS for 5 min.</li>
	<li>Observe the slide under a light microscope using 40X dry lens to ensure that the slides are digested properly and no cytoplasm is present and the chromosome morphology is preserved. Select an area for hybridization using a diamond pen.</li>
</ol>
3. Chromosome and probe denaturation and hybridization
<ol>
	<li>Prepare fresh denaturing solution (70% formamide/2X SSC, pH 7.0) and prewarm to 70-80°C in a coplin jar placed in a water bath Place the slide in the coplin jar containing the denaturing solution in a water bath at 70°C for mouse chromosomes and 80°C for human chromosomes for 30s-1.5 min.</li>
	<li>Immediately place the slide in ice-cold 70% ethanol for 3 min followed by 80% and 100% ethanol for 3 min each and air dry. Examine the slide for chromosome morphology. Good chromosome morphology is denoted by dark chromosomes and not "phase-light" or halo chromosomes.</li>
	<li>Warm the SkyPaint probe (SKY paint kit; vial#1) at 37°C with shaking for 20 min, vortex and centrifuge briefly at 1000 r.p.m. for a few seconds.</li>
	<li>Denature the probe in a thermocycler programmed for a two-step cycle at 85°C for 5 min cycle followed by 37°C for 60 min to allow labeled-probe DNA for preannealing.</li>
	<li>Apply 10μl of the denatured probe onto the area of hybridization and cover with a 22 mm x 40 mm coverglass making sure not to trap air bubbles. Seal the edges of the coverglass with rubber cement and incubate in a humidified chamber at 37°C for 48-72 hrs.</li>
</ol>
4. Fluorescent probe detection
<ol>
	<li>Remove the coverglass carefully and place the slide in a coplin jar containing prewarmed (45°C) washing solution I (freshly prepared 50% formamide in 2X SSC). Wash for 5 min three times at 45°C in a shaking water bath at 45 r.p.m.</li>
	<li>Wash the slide in washing solution II (1X SSC) at 45°C for 5 min twice with shaking.</li>
	<li>Wash the slide in washing solution III (4X SSC/0.1% Tween 20) for 5 min at 45°C with shaking.</li>
	<li>Apply 80 μl of blocking reagent (SKY paint kit; vial#2), place a coverslip and incubate at 37°C for 30 min.</li>
	<li>Remove the slide and allow the fluid to drain. Apply 80 μl of Cy5 staining reagent (Concentrated Antibody Detection CAD kit; vial#3), apply a coverglass and incubate at 37°C for 40 min.</li>
	<li>Wash the slide with washing solution III at 45°C for 5 min three times with shaking.</li>
	<li>Apply 80 μl of Cy5.5 staining reagent (Concentrated Antibody Detection CAD kit; vial#4), place a coverglass and incubate at 37°C for 40 min.</li>
	<li>Wash the slide with washing solution III at 45°C for 5 min three times with shaking.</li>
	<li>Tilt the slide and allow the fluid to drain. Apply 20 μl of the anti-fade DAPI reagent (SKY paint kit; vial#5) and place a 24 mm x 60 mm microscope coverglass. Carefully remove air bubbles that might have formed. Slides can be imaged immediately or stored at 4°C in the dark for no longer than 1 week.</li>
</ol>
5. Image acquisition and analysis
<ol>
	<li>Image acquisition is accomplished by viewing metaphase slides using an Olympus microscope equipped with a 60X oil immersion lens, a Spectral cube (custom designed triple band pass filter), a DAPI filter and a sagnac interferometer module with a CCD camera.</li>
	<li>Spectral-Karyotypes were carried out using SKY View software (Applied spectral imaging Version 1.62) following the user's manual.</li>
	<li>After analyzing the images, the chromosomes can be viewed as color images (with specific fluorescent colors), pseudo color images (with colors for classification) and inverted DAPI images (specific banding pattern).</li>
</ol>
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).
<img alt="Fluorescence in situ hybridization process diagram; chromosome denaturation, hybridization, analysis." src="/files/ftp_upload/3887/3887fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
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.
<img alt="Chromosome analysis, spectral karyotyping diagrams, wild-type vs. mutant, mouse, human, ADPKD." src="/files/ftp_upload/3887/3887fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
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.

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No conflicts of interest declared.

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><tbody><tr><td>DMEM</td><td>Cellgro</td><td>10-013-CV</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Hyclone</td><td>SH30088-03</td><td></td></tr><tr><td>Penecillin/Streptomycin</td><td>Thermo Fisher Scientific, Inc.</td><td>SV30010</td><td></td></tr><tr><td>Colcemid</td><td>Roche Group</td><td>10 295 892 001</td><td>10 &amp;#x03BC;g/ml</td></tr><tr><td>HCl</td><td>Fisher Scientific</td><td>A144-500</td><td></td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>S77375-1</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Thermo Fisher Scientific, Inc.</td><td>SH30256-01</td><td></td></tr><tr><td>SKY paint probe kit (Human)</td><td>Applied Spectral Imaging</td><td>SKY000028</td><td></td></tr><tr><td>SKY paint probe kit (Mouse)</td><td>Applied Spectral Imaging</td><td>SKY000030</td><td></td></tr><tr><td>Concentrated antibody detection kit</td><td>Applied Spectral Imaging</td><td>SKY000033</td><td></td></tr><tr><td>Trypsin</td><td>Thermo Fisher Scientific, Inc.</td><td>SH30236.01</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A433P-4</td><td></td></tr><tr><td>Acetic acid</td><td>Fisher Scientific</td><td>A38-212</td><td></td></tr><tr><td>RNase A</td><td>Roche Group</td><td>10 109 169 001</td><td></td></tr><tr><td>Pepsin</td><td>Sigma-Aldrich</td><td>P6887-5G</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fluka</td><td>63069-500ML</td><td></td></tr><tr><td>37% Formaldehyde</td><td>Mallinckrodt Baker Inc.</td><td>2106-02</td><td></td></tr><tr><td>20X SSC</td><td>Promega Corp.</td><td>V4261</td><td></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></td></tr><tr><td>Microscope glass slides</td><td>Fisher Scientific</td><td>12-549</td><td></td></tr><tr><td>Microscope cover glass 24x60mm</td><td>VWR international</td><td>16004-312</td><td></td></tr><tr><td>Rubber cement</td><td>Elmer&amp;rsquo;s</td><td></td><td></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></td></tr><tr><td>Shaking platform/Orbital shaker</td><td>Bellco Glass</td><td></td><td></td></tr><tr><td>Shaking/water bath</td><td>Precision Scientific</td><td></td><td></td></tr><tr><td>DAPI filter cube</td><td>Chroma Technology Corp.</td><td></td><td></td></tr><tr><td>SKY filter cube</td><td>Chroma Technology Corp.</td><td></td><td></td></tr><tr><td>SpectraCube</td><td>Applied Spectral Imaging</td><td></td><td></td></tr><tr><td>Inverted cell culture microscope</td><td>Nikon Instruments</td><td>Nikon Eclipse TS100</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Olympus Corporation</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 ...

spectral-karyotyping-to-study-chromosome-abnormalities-humans-mice

Research

JoVE Journal

Medicine

38.3K Views.  University of Toledo, College of Pharmacy and Pharmaceutical Sciences. Spectral Karyotypering (SKY) is een geavanceerde cytogenetica techniek om genomische en chromosomale afwijkingen te identificeren. Deze techniek profiteert van chromosoom schilderkunst probes, waarmee indeling van alle chromosomen. SKY kan ook worden aangegeven complexe chromosoomafwijkingen en de segregatie defecten in muizen en mensen met diverse ziekten, waaronder polycystische nierziekte.

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

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.

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.

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

Genetics

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

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.

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

Immunology and Infection

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

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.

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

Biology

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays.

Ion channels expressed in renal tubular epithelium play a significant role in the pathology of polycystic kidney disease. Here we describe experimental protocols used to perform patch-clamp analysis and intracellular calcium level measurements in cystic epithelium freshly isolated from rodent kidneys.

Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal ...

Use of 3D Robotic Ultrasound for In Vivo Analysis of Mouse Kidneys

Common modalities for in vivo imaging of rodents include positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Each method has limitations and advantages, including availability, ease of use, cost, size, and the use of ionizing radiation or magnetic fields. This protocol describes the use of 3D robotic US for in vivo imaging of rodent kidneys and heart, subsequent data analysis, and possible research applications. Practical applications of robotic US are the quantification of total kidney volume (TKV), as well as the measurement of cysts, tumors, and vasculature. Although the resolution is not as high as other modalities, robotic US allows for more practical high throughput data collection. Furthermore, using US M-mode imaging, cardiac function may be quantified. Since the kidneys receive 20%-25% of the cardiac output, assessing cardiac function is critical to the understanding of kidney physiology and pathophysiology.

Use of 3D Robotic Ultrasound for In Vivo Analysis of Mouse Kidneys

This protocol demonstrates robotic ultrasound (US) as a practical, cost-effective, and quick alternative to traditional non-invasive image modalities.

Common modalities for in vivo imaging of rodents include positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), ...

Cytogenetics

Cytogenetics is the field of study devoted to chromosomes, and involves the direct observation of a cell&#8217;s chromosomal number and structure, together known as its karyotype. Many chromosomal abnormalities are associated with disease. Each chromosome in a karyotype can be stained with a variety of dyes to give unique banding patterns. More recent techniques, including comparative genomic hybridization and fluorescence in situ hybridization (FISH), allow for detecting specific chromosomal features or abnormalities.This video will begin by examining the principles of these classical and modern cytogenetics techniques. This is followed by an examination of a general protocol for performing FISH. Finally, several examples of how karyotyping can be applied to various medical applications are presented.

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

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