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 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 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 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. 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'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.
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.
1. Chromosome Harvesting of Adherent Cells
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 °C).
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.
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.
Figure 1. A successful chromosomal spread in which the chromosomes are of average length, well spread and easily discernible from one another, and sufficient contrast between light and dark bands. Good chromosome morphology allows for the identification of individual chromosomes and evaluation for any rearrangements indicative of chromosome instability.
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 Colcemid incubation time. Insufficient time in Colcemid yields fewer metaphase spreads and longer, overlapped chromosomes. Longer incubation times in Colcemid will result in shorter and thicker chromosomes which are difficult to analyze. Another important variable is the molarity of the hypotonic solution. A 0.075 M potassium chloride solution will swell the cells just enough to yield proper chromosome spreading, without lysing the cells. While adding the Carnoy's Fixative solution, the first 5 ml must be added to the pellet while mixing. This ensures that all of the extra proteins are removed before storing the samples or preparing slides. If in the case that the cells are not mixed well enough, a yellow protein cap will form on top of the pellet, which can cause complications during subsequent procedures.
Proper slide preparation is essential. While dropping the resuspended cell pellet onto slides, make sure that the slide is tilted to approximately 45° angle and there is enough distance (at least 2 inches) from the dropper to the slide so that the chromosomes can properly disperse onto the slide for analysis. Flooding the slide with fixative immediately following dropping the cells on to the slide will also help chromosomes to spread. Temperature and humidity, which affect how fast the cell suspension dries on to the slide, are other factors which affect chromosome spreading. These can be controlled by preparing slides in an environment with approximately 50% humidity and a temperature of about 20-25 °C, and/or by placing slides on a slide warmer for faster drying. For G-Banding, the main factor that affects the quality of the chromosomes is the trypsin exposure times. With a longer trypsin exposure, chromosomes may appear diffused and swollen. Conversely, an inadequately short trypsin incubation will yield chromosomes with indistinguishable bands and little contrast. The trypsin incubation timing is subject to change depending on each specific cell line and harvesting conditions. Therefore, a representative G-banded slide should be first prepared to evaluate the trypsin conditions before staining the rest of the slides. Fetal bovine serum is used to inactivate the trypsin activity prior to staining. We provide the protocol using Giemsa (GTG), but Wright's stain may be used instead of Giemsa for GTW banding and yields similar results.
This procedure is relatively inexpensive and effective to perform. G-banding can be used to diagnose chromosome abnormalities such as translocations, deletions, and aneuploidy which are commonly seen in malignancies, genetic disorders, and stem cells cultured in vitro10,11,13. This provides the indispensable visualization of the chromosome constitution and the global evaluation of the entire genome in multiple cells. It is commonly used in clinical and research laboratories worldwide for the diagnosis, prognosis, and therapeutic evaluation of cancer cells10. Prenatal and postnatal tissue samples are routinely evaluated for the identification of numerical and structural abnormalities which cause genetic disorders such as Down syndrome. Stem cells can be rapidly evaluated for the presence of chromosome instability. However, the resolution of G-banding is limited for the identification of microdeletions or complex chromosome abnormalities as seen in metastatic malignancies.
Therefore, when routine chromosome G-banding is supplemented with procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY), detection rates of chromosomal abnormalities are increased dramatically23. For this reason, the utilization of the present protocol in combination with these other molecular cytogenetic procedures is increasingly being used by different laboratories for the evaluation of chromosome instability in both the clinical and research setting13-15.
The authors have nothing to disclose.
Work described in this manuscript was made possible by funding from the Patrick F. Taylor Foundation.
KaryoMAX Colcemid | Gibco | 15212-012 | |
HBSS Buffer | Gibco | 24020-117 | |
0.5% Trypsin EDTA 10x | Gibco | 15400-054 | |
Permount | Fisher Scientific | SP15-100 | |
Buffer Tablets "GURR" | Gibco | 10580-013 | |
Geimsa Stain | Ricca Chemical Company | 3250-16 | |
Centrifuge 5810 | Eppendorf | ||
Methanol | Caledon Laboratory Chemicals | 6700130 | |
Glacial Acetic Acid | Krackeler Scientific Inc. | 11-9508-05 |