Work described in this manuscript was made possible by funding from the Patrick F. Taylor Foundation.

1. Chromosome Harvesting of Adherent Cells
<ol>
	<li>Standard Protocol
	<ol>
		<li>Grow cells according to specific cell culturing conditions. When the cells have reached logarithmic phase (80% confluency), add 10 &#956;l/ml of Colcemid to the cell culture flask. A minimum of 2 x 106 cells is recommended.</li>
		<li>Incubate cells at 37 &#176;C in a 5% CO2 incubator for 45 min. Using a sterile pipette, transfer media from cells into a 15 ml conical tube. Set aside.</li>
		<li>Gently wash the cells by adding 2 ml of HBSS Buffer into the flask. Swirl buffer and then remove using a pipette. Discard.</li>
		<li>Add 1 ml of trypsin, ensuring that it covers the entire surface of the flask. Only leave the cells in trypsin for about 2 min. Once the majority of the cells have detached, pipette the media in the conical tube back onto the cells.</li>
		<li>Transfer the cell suspension in 10 ml aliquots into 15 ml conical tubes. Centrifuge at 200 x g for 10 min. Remove supernatant and resuspend the pellet.</li>
		<li>Add 10 ml of 0.075 M KCl which has been prewarmed to 37 &#176;C to the remaining pellet in the conical tube. Vortex tube at medium speed to mix KCl and cells.</li>
		<li>Incubate cells at 37 &#176;C for 10 min. Centrifuge at 200 x g for 5 min at 25 &#176;C. Remove supernatant (until about 0.5 ml remains) and resuspend pellet.</li>
		<li>Carefully add 5 ml of fresh Carnoy's Fixative (3:1 ratio of methanol:glacial acetic acid) to the cells while vortexing. Then add 5 ml more of fixative without vortexing for a total of 10 ml.</li>
		<li>Centrifuge at 200 x g for 5 min. Remove supernatant and resuspend cells. Add 5 ml of fixative to each tube.</li>
		<li>Centrifuge at 200 x g for 5 min. Remove supernatant and resuspend cells. Add 5 ml of fixative to each tube. The cells can now be stored in 4 &#176;C for up to one year.</li>
	</ol>
	</li>
	<li>Modification to Protocol: Chromosome harvesting of lymphoblastoid cell lines
	<ol>
		<li>Grow cells according to specific cell culturing conditions. (Use flask appropriate for the amount of cells being cultured). When the cells have reached logarithmic phase (approximately 2 days after cells are split), add 10 &#956;l/ml of Colcemid to the cell culture flask.</li>
	</ol>
	</li>
	<li>Modification to Protocol: Chromosome harvesting from whole blood 
	Phytohemagglutinin (PHA), a lectin derived from the red kidney bean, is a powerful mitogen for human T-cells16. 72 hr after the addition of PHA to the culture, about 45% of cells are in S phase. This represents the peak mitotic activity, and is the optimum point at which to harvest for chromosome studies. Other mitogens such as pokeweed (1-10 &#956;g/ml) may be used when analyzing B cells17,18.
	<ol>
		<li>Collect at least 1 ml of whole blood in a green top sodium heparin tube. Use within 3 days after collection. Store blood at RT until ready to use.</li>
		<li>Aliquot 0.25 ml of whole blood in 10 ml of complete RPMI media containing L-glutamine (20% fetal bovine serum, 1% Penicillin/streptomycin, 1% fungizone, and 1% PHA). Culture at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
	<li>Modification to Protocol: Chromosome harvesting from bone marrow Cytogenetic analysis on bone marrow is helpful in many malignant hematologic disorders as the observation of a chromosomally abnormal clone may be diagnostic for a specific type of leukemia. Chromosome studies are also used to assess disease progression such as the onset of blast crisis and response to treatment. Since bone marrow cells are actively dividing, no mitogen stimulation is necessary9,19. For acute leukemias 24 and 48 hr cultures are also set up.
	<ol>
		<li>Collect at least 1 ml of bone marrow in a green top sodium heparin tube. Aliquot 0.25 ml of bone marrow in 10 ml of complete RPMI media containing L-glutamine (20% fetal bovine serum, 1% Penicillin/streptomycin, 1% fungizone). Culture at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
</ol>
2. Slide Preparation and Solid Staining
A rapid evaluation of one representative slide will provide information on the quality of the harvest before continuing to further procedures such as G-banding, FISH, CGH, or SKY. Some laboratories prefer to solid stain the cells for rapid harvesting and chromosome assessment. Alternatively, a phase contrast microscope may be used for this analysis. Slides are best prepared when the humidity is approximately 50% and the temperature ambient (20-25 &#176;C).
<ol>
	<li>Centrifuge the cells at 200 x g for 5 min at 25 &#176;C. Remove the supernatant until only 0.3-0.5 ml remains.</li>
	<li>After gently resuspending the pellet, pipette three drops of the cell suspension from a distance of about 2 in onto a slide which is tilted at an angle of about 45&#176; and allow the suspension to roll across the slide. Add one large drop of fresh Carnoy's Fixative to the slide.</li>
	<li>Dry the back of the slide on a paper towel and then sit the slide out to dry for at least 10 min. The slide should be completely dry.</li>
	<li>Prepare fresh Giemsa Staining Solution (3:1 ratio of Gurr Buffer and Giemsa Stain). Place the slides on a staining rack. Cover the entire slide in the Giemsa staining solution. Let the slides remain in the staining solution for 5 min. Rinse slides with distilled water, drain, and allow to air dry.</li>
	<li>Add 4 drops of Permount and a cover slip to the slide. Make sure there are no bubbles under the coverslip. The excess Permount can be removed with a paper towel.</li>
	<li>Analyze cells with a light microscope under 10X and 100X magnification. If the metaphase cells are abundant and well spread, the remaining slides can be used for other experimental procedures.</li>
</ol>
3. G-Banding Using Trypsin and Giemsa (GTG)
Trypsin is a proteolytic enzyme which denatures euchromatic histones in DNA regions with higher transcriptional activity resulting. Following Giemsa staining, these regions will appear as light bands. Highly condensed chromatin with little or no transcriptional activity (heterochromatin) will have a large portion of its histones protected from the trypsin and will therefore stain darkly following Giemsa staining. It is essential to initially G-band one slide to monitor the conditions and adjust the trypsin timing if necessary for the subsequent slides.
<ol>
	<li>Add the following solutions to 4 Coplin jars. Jar #1= 30 ml of 1x HBSS and 4 ml of 10x trypsin (0.5%) 
	Jar #2= 50 ml of 1x HBSS 
	Jar #3= 45 ml of 1x HBSS and 5 ml of fetal bovine serum 
	Jar #4= 50 ml of 1x HBSS</li>
	<li>Immerse each slide in Jar #1 for 5 sec, quick rinse in Jar #2, leave each slide in Jar #3 for at least 30 sec, quick rinse in Jar #4 then allow slides to dry.</li>
	<li>Prepare fresh Giemsa Staining Solution (3:1 ratio of Gurr Buffer and Giemsa Stain). Place the slides on a staining rack. Cover the entire slide in the Giemsa staining solution. Let the slides remain in the staining solution for 5 min.</li>
	<li>Rinse slides in distilled water in the same order that they were stained. Allow the slides to dry for about 10 min. The slide should be completely dry.</li>
	<li>Add 4 drops of Permount and a cover slip to the slide. Make sure there are no bubbles under the coverslip. The excess Permount can be removed with a paper towel.</li>
	<li>Analyze cells with a light microscope under 10X and 100X magnification. Adjust trypsin timing of the rest of the slides based on the results of the first slide.</li>
</ol>

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The authors have nothing to disclose.

We have utilized the present procedure for chromosome isolation from cells of various organisms, including various cell types obtained from human, Rhesus macaques, rats, and mice11,20-22. The standard protocol is provided, but certain key steps and variables may need to be adjusted for these diverse types of cells. Several specific steps are crucial in both the chromosomal preparation and G-banding to ensure the best possible quality of the results. One of the variables which can affect the assay is the Colcem...

Chromosome analysis is a conventional technique utilized worldwide to diagnose chromosome instability and rearrangements leading to genetic disorders and malignancy1,2,8,9. In addition, a higher resolution for the diagnosis and research of constitutional and cancer-acquired genetic abnormalities can be achieved with the combination of the classical cytogenetic procedures and molecular cytogenetic methodologies such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15. More recently, these techniques have been utilized for the evaluation of chromosome instability associated with stem cell research. Karyotypic abnormalities such as aneuploidy of long-term cultured embryonic cells (ES) and adult stem cells of various organisms have been reported by multiple laboratories. Recent evidence supports that some cell lines are inherently more inclined to chromosome instability regardless of culture conditions. For this reason, when establishing and/or maintaining human, mouse, or Rhesus stem cell lines, chromosome analysis is recommended as part of the quality control process. Many reports describe the increasing interest in the use of routine and molecular cytogenetics to monitor the chromosomal stability of stem cells and malignant cells or various organisms in culture10-13. These protocols are increasingly being used by nongenetic laboratories for rapid chromosomal assessment of their cultured cells13. We present our basic procedures for chromosome preparation from various cell types, which can be applied for both clinical and research purposes and cells derived from various organisms.

<table><tbody><tr><td>KaryoMAX Colcemid</td><td>Gibco</td><td>15212-012</td><td></td></tr><tr><td>HBSS Buffer</td><td>Gibco</td><td>24020-117</td><td></td></tr><tr><td>0.5% Trypsin EDTA 10x</td><td>Gibco</td><td>15400-054</td><td></td></tr><tr><td>Permount</td><td>Fisher Scientific</td><td>SP15-100</td><td></td></tr><tr><td>Buffer Tablets &quot;GURR&quot;</td><td>Gibco</td><td>10580-013</td><td></td></tr><tr><td>Geimsa Stain</td><td>Ricca Chemical Company</td><td>3250-16</td><td></td></tr><tr><td>Centrifuge 5810</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Methanol</td><td>Caledon Laboratory Chemicals</td><td>6700130</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Krackeler Scientific Inc.</td><td>11-9508-05</td><td></td></tr></tbody></table>

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.

High quality metaphase spreads are essential for chromosome analysis. A successful assay yields chromosomes which are well spread and of suitable chromosome morphology. Properly G-banded chromosomes contain the characteristic light and dark banding patterns.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50203/50203fig1highres.jpg" src="/files/ftp_upload/50203/50203fig1.jpg" /> 
Figure 1.&#160;A successfu...

Watch this Scientific Journal Video about Chromosome Preparation From Cultured Cells at JoVE.com

Chromosome Preparation From Cultured Cells

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 ...

chromosome-preparation-from-cultured-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

80.8K Views.  Louisiana State University Health Science Center. 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).

Video: Chromosome Preparation From Cultured Cells

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. To assess the impact of PM on nuclear genetic integrity, structural chromosomal aberrations are scored in the metaphase spreads of mouse RAW264.7 macrophage cells. PM is collected from ambient air with a high volume total suspended particles sampler. The collected material is solubilized and filtered to retain the water-soluble, fine portion. The particles are characterized for chemical composition by nuclear magnetic resonance (NMR) spectroscopy. Different concentrations of particle suspension are added onto an in vitro culture of RAW264.7 mouse macrophages for a total exposure time of 72 hr, along with untreated control cells. At the end of exposure, the culture is treated with colcemid to arrest cells in metaphase. Cells are then harvested, treated with hypotonic solution, fixed in acetomethanol, dropped onto glass slides and finally stained with Giemsa solution. Slides are examined to assess the structural chromosomal aberrations (CAs) in metaphase spreads at 1,000X magnification using a bright-field microscope. 50 to 100 metaphase spread are scored for each treatment group. This technique is adapted for the detection of structural chromosomal aberrations (CAs), such as chromatid-type breaks, chromatid-type exchanges, acentric fragments, dicentric and ring chromosomes, double minutes, endoreduplication, and Robertsonian translocations in vitro after exposure to PM. It is a powerful method to associate a well-established cytogenetic endpoint to epigenetic alterations.

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter.

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

Genetics

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein interactions in yeast. It allows analysis of a target region of interest without the need for prior knowledge about likely proteins bound to the target region. This, in theory, allows HyCCAPP to be used to analyze any genomic region of interest, and it provides sufficient flexibility to work in different cell systems. This method is not meant to study binding sites of known transcription factors, a task better suited for Chromatin Immunoprecipitation (ChIP) and ChIP-like methods. The strength of HyCCAPP lies in its ability to explore DNA regions for which there is limited or no knowledge about the proteins bound to it. It can also be a convenient method to avoid biases (present in ChIP-like methods) introduced by protein-based chromatin enrichment using antibodies. Potentially, HyCCAPP can be a powerful tool to uncover truly novel DNA-protein interactions. To date, the technology has been predominantly applied to yeast cells or to high copy repeat sequences in mammalian cells. In order to become the powerful tool we envision, HyCCAPP approaches need to be optimized to efficiently capture single-copy loci in mammalian cells. Here, we present our adaptation of the initial yeast HyCCAPP capture protocol to human cell lines, and show that single-copy chromatin regions can be efficiently isolated with this modified protocol.

This is a method to identify novel DNA-interacting proteins at specific target loci, relying on sequence-specific capture of crosslinked chromatin for subsequent proteomic analyses. No prior knowledge about potential binding proteins, nor cell modifications are required. Initially developed for yeast, the technology has now been adapted for mammalian cells.

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to be responsible for carcinogenesis in such tissues. Although proliferative polyploid cells are requisite for analyzing chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is rather challenging. Induction of tetraploidy by chemical agents usually results in a mixture of diploid and tetraploid populations, and most studies employed fluorescence-activated cell sorting or cloning by limiting dilution to separate tetraploid from diploid cells. However, these procedures are time-consuming and laborious. The present report describes a relatively simple protocol to induce proliferative tetraploid cells from normal human fibroblasts with minimum contamination by diploid cells. Briefly, the protocol is comprised of the following steps: arresting cells in mitosis by demecolcine (DC), collecting mitotic cells after shaking off, incubating collected cells with DC for an additional 3 days, and incubating cells in drug-free medium (They resume proliferation as tetraploid cells within several days). Depending on cell type, the collection of mitotic cells by shaking off might be omitted. This protocol provides a simple and feasible method to establish proliferative tetraploid cells from normal human fibroblasts. Tetraploid cells established by this method could be a useful model for studying chromosome instability and the oncogenic potential of polyploid human cells.

Although proliferative polyploid cells are necessary to analyze chromosomal instability of polyploid cells, creating such cells from nontransformed human cells is not easy. The present report describes relatively simple procedures to establish proliferative tetraploid cells free of a diploid population from normal human fibroblasts.

Polyploid (mostly tetraploid) cells are often observed in preneoplastic lesions of human tissues and their chromosomal instability has been considered to ...

Developmental Biology

Capturing Chromosome Conformation Across Length Scales

Chromosome conformation capture (3C) is used to detect three-dimensional chromatin interactions. Typically, chemical crosslinking with formaldehyde (FA) is used to fix chromatin interactions. Then, chromatin digestion with a restriction enzyme and subsequent religation of fragment ends converts three-dimensional (3D) proximity into unique ligation products. Finally, after reversal of crosslinks, protein removal, and DNA isolation, DNA is sheared and prepared for high-throughput sequencing. The frequency of proximity ligation of pairs of loci is a measure of the frequency of their colocalization in three-dimensional space in a cell population.
A sequenced Hi-C library provides genome-wide information on interaction frequencies between all pairs of loci. The resolution and precision of Hi-C relies on efficient crosslinking that maintains chromatin contacts and frequent and uniform fragmentation of the chromatin. This paper describes an improved in situ Hi-C protocol, Hi-C 3.0, that increases the efficiency of crosslinking by combining two crosslinkers (formaldehyde [FA] and disuccinimidyl glutarate [DSG]), followed by finer digestion using two restriction enzymes (DpnII and DdeI). Hi-C 3.0 is a single protocol for the accurate quantification of genome folding features at smaller scales such as loops and topologically associating domains (TADs), as well as features at larger nucleus-wide scales such as compartments.

Hi-C 3.0 is an improved Hi-C protocol that combines formaldehyde and disuccinimidyl glutarate crosslinkers with a cocktail of DpnII and DdeI restriction enzymes to increase the signal-to-noise ratio and the resolution of chromatin interaction detection.

Chromosome conformation capture (3C) is used to detect three-dimensional chromatin interactions. Typically, chemical crosslinking with formaldehyde (FA) ...

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

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high quality plant chromosome spreads is challenging due to the rigid cell wall. One of the approved methods for the preparation of plant chromosomes is a so-called drop preparation, also known as drop-spreading or air-drying technique. Here, we present a protocol for the fast preparation of mitotic chromosome spreads suitable for the FISH detection of single and high copy DNA probes. This method is an improved variant of the air-dry drop method performed under a relative humidity of 50%-55%. This protocol comprises a reduced number of washing steps making its application easy, efficient and reproducible. Obvious benefits of this approach are well-spread, undamaged and numerous metaphase chromosomes serving as a perfect prerequisite for successful FISH analysis. Using this protocol we obtained high-quality chromosome spreads and reproducible FISH results for Hordeum vulgare, H. bulbosum, H. marinum, H. murinum, H. pubiflorum and Secale cereale.

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

Chromosome Preparation From Cultured Cells

Summary

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Chapters in this video

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

Establishment of Proliferative Tetraploid Cells from Nontransformed Human Fibroblasts

Capturing Chromosome Conformation Across Length Scales

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants