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.
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Method Article
Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH
In This Article
Erratum Notice
Important: There has been an erratum issued for this article. View Erratum Notice
Summary
Abstract
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.
Introduction
Cancer is caused by the accumulation of mutations affecting genes that regulate normal cell growth. Mutation is a consequence of changes to the structure and sequence of the genome caused by damage to DNA. DNA damage can occur through a variety of process, including exogenous agents such as ionizing radiation, and as a by-product of normal cellular metabolism, such as spontaneous deamination of nucleotide bases or damage occurring by contact with reactive oxygen species1.
Although mammalian cells possess a range of repair activities which can reverse DNA damage or restore the sequence at break sites, mutations nonetheless accumul....
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Protocol
This procedure was approved by the Institutional Animal Care and Use Committee at Rutgers, the State University of New Jersey. Mice were treated in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Scientists should consult their national and institutional animal organizations for established and approved guidelines.
Before beginning, prepare the solutions listed in Table 1.
1. B Cell Isolation and Activation
Euthanize a mouse using CO2 followed by cervical dislocation. Position the mouse so the left side of the body is up and spray the....
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Results
Metaphase chromosomes derived from one cell should form a discrete cluster containing 40 chromosomes (if using mouse cells) (Figure 1A). Each chromosome has a centromere at one end, which is visible as a light blue sphere. In normal chromosomes, two telomere signals are seen at the end of the chromatids and two signals by each centromere. Interphase nuclei will be present on the slide as well. These will be visible as blue spheres, with red telomere signals, but no condensed chromosomes (see for example .......
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Discussion
Whereas activated B lymphocytes are particularly suited to preparation of mitotic chromosome spreads, other cell types can also be used. T lymphocytes share many of the advantages of B cells, as they can be purified from the spleen or lymph nodes, and have a high mitotic index when stimulated by growth in medium containing appropriate mitogens8. Embryonic Stem cells (ES cells) are also suited for metaphase chromosome analysis9. If these are not available, fibroblast cells can be used, although a lon.......
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Disclosures
The authors have no conflicting interests.
Acknowledgements
This work is supported by NIH grant R00 CA160574 (SFB) and by a fellowship of the Rutgers Biotechnology Training Program (to SMM).
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| 50% Dextran sulfate | Intergen | S4030 | |
| Deionized formamide | Ambion | 9342 | |
| Pepsin (5 g) | Sigma | P 6887 | |
| FCS | Gemini | 100-106 | |
| LPS (100 mg) | Sigma | L2630 | |
| IL-4 (5 μg) | Sigma | I1020 | |
| PNA Telomere Probe | PNA Bio Inc | F1002 | (CCCTAACCCTAACCCTAA) |
| Colcemid | Roche | 295892 | |
| Mowiol 4-88 | Sigma | 81381-50g | |
| CD43 (ly-48_) micro-beads | Miltenyl Biotec. | 130-049-801 | |
| DAPI | Sigma | 32670-5MG | |
| Eclipse E800 | Nikon | ||
| AxioImager.Z2 | Zeiss | ||
| CDS-5 Cytogenetic Drying Chamber | Thermotron |
References
- Sancar, A., Lindsey-Boltz, L. A., Unsal-Kacmaz, K., Linn, S. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Ann Rev Biochem. 73, 39-85 (2004).
- Sperka, T., Wang, J., Rudolph, K. L. DNA damage checkpoints in stem cells, ageing and cancer.....
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Reprints and Permissions
Erratum
Formal Correction: Erratum: Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH
Posted by JoVE Editors on 1/01/1970. Citeable Link.
A correction was made to: Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH. Table 1 (the list of solutions to prepare) was omitted from the Protocol section. The contents of Table 1 are as follows:
| Solution | Reagents | Comments | Storage |
| B Cell Wash Buffer | 500 ml 1x Hanks Balanced Salt Solution | Filter through 0.22 μm stericup filtration unit | 4 °C |
| 5 ml 50 °C heat inactivated FCS | |||
| 5 ml 100x Pen/Strep | |||
| B Cell Medium | 418.5 ml RPMI-1640 | Filter through 0.22 μm stericup filtration unit | 4 °C |
| 50 ml 50 °C heat inactivated FCS | |||
| 5 ml 100x Pen/Strep | |||
| 5 ml glutamine | |||
| 5 ml nonessential amino acids | |||
| 5 ml sodium pyruvate | |||
| 1.8 ml 14.2 M 2-mercaptoethanol | |||
| 5 ml 1M HEPES | |||
| Fixative | 3:1 v/v methanol/glacial acetic acid | Make fresh | |
| 0.075 M KCl | 1.395 g KCl | Room Temperature | |
| 250 ml distilled H2O | |||
| FISH Master Mix | 40 ml 50% Dextran sulfate | Vortex the solution and leave in a shaking platform overnight | Aliquot and store at -20 °C |
| 20 ml 20x SSC | |||
| 40 ml sterile distilled H2O | |||
| 70% Formamide/2x SSC | 10 ml 20x SSC | Adjust to pH 7 with 1M HCl | Aliquot and store at -20 °C |
| 20 ml distilled H2O | |||
| 70 ml deionized formamide | |||
| 0.01 M HCl | 1 ml 1M HCl | Adjust to pH 2.0 | Room Temperature |
| 99 ml distilled H2O | |||
| 1x PBS/MgCl2 | 50 ml 1M MgCl2 | Room Temperature | |
| 950 ml 1x PBS | |||
| 50% Formamide/2x SSC | 20 ml 20x SSC | Adjust to pH 7.25 Formamide used in this step does not have to be deionized. | Make fresh |
| 80 ml distilled H2O | |||
| 100 ml formamide | |||
| 4x SSC/0.1% Tween20 | 50 ml 20x SSC | Room Temperature | |
| 200 ml distilled H2O | |||
| 250 μl Tween20 | |||
| DAPI staining solution (80 ng/ml) | 40 μl 0.2mg/ml DAPI stock | 4 °C in light protected staining jar | |
| 100 ml 2x SSC | |||
| Mowiol Mounting Medium | 6 g glycerol | Incubate at 50 °C with stirring for 2hrs | Aliquot and store at 4 °C |
| 2.4 g Mowiol 4-88 | |||
| 6 ml distilled H2O | |||
| 12 ml 0.2 M TrisCl pH 8.5 | |||
| 230 μl 1% Thimerosal (w/v in water) |
Table 1. List of solutions.
