Assays for Detecting Chromosomal Aberrations

Catherine B. Klein1, Limor Broday1, Max Costa1

1 New York University school of Medicine, New York, New York
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 3.7
DOI:  10.1002/0471140856.tx0307s03
Online Posting Date:  May, 2001
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Abstract

This unit describes the techniques used to investigate chromosome structure to detect changes induced by toxicant exposure. Protocols are included for detecting microcucleus (MN) formation, changes in banding patterns by Giemsa banding or fluorescence in situ hybridization (FISH) using chromosome‚Äźspecific probes, and sister chromatid exchange (SCE) in cultures of established cell lines.

     
 
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Table of Contents

  • Basic Protocol 1: Micronucleus Assay for Chromosomal Aberrations
  • Basic Protocol 2: Giemsa or Giemsa/trypsin Staining to Evaluate Chromosome Aberrations
  • Basic Protocol 3: Evaluation of Chromosome Aberrations by Painting with Fluorescence In Situ Hybridization (FISH) Probes
  • Support Protocol 1: Preparation of Metaphase Chromosome Spreads
  • Basic Protocol 4: Detection of Sister Chromatid Exchanges
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Micronucleus Assay for Chromosomal Aberrations

  Materials
  • Cells to be tested, in exponential growth phase or PHA‐stimulated lymphocytes
  • Medium and supplements appropriate for the cell line studied ( appendix 3B)
  • Agent to be tested for toxicity, in appropriate solvent
  • 1 mg/ml cytochalasin B (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Hypotonic solution: 0.075 M KCl
  • 80% to 100% ice‐cold methanol, freshly prepared
  • 5% (w/v) Giemsa solution (prepare fresh): dilute Giemsa stain (see recipe) 1:19 (v/v) in Sorenson's phosphate buffer, pH 6.8 (see recipe)
  • 60‐mm petri dishes
  • Microscope slides, −20°C
  • Additional reagents and equipment for culturing and trypsinizing cells ( appendix 3B), mutagenesis assays in cells (unit 3.3), and slide preparation by dropping (see protocol 4) or cytospinning (unit 2.2)

Basic Protocol 2: Giemsa or Giemsa/trypsin Staining to Evaluate Chromosome Aberrations

  Materials
  • Slides containing metaphase spreads (see protocol 4) from treated and control cells
  • 10% (w/v) Giemsa solution (for Giemsa staining) or 5% (w/v) Giemsa solution (for trypsin/Giemsa staining): dilute Giemsa stain (see recipe) 1:9 (for 10%) or 1:19 (for 5%) in Sorenson's phosphate buffer, pH 6.8 (see recipe), prepare diluted stains fresh
  • 0.05% (w/v) trypsin: dilute 2.5% (w/v) stock in PBS or other physiological saline solution
  • PBS ( appendix 2A) or saline solution
  • Permount (Fisher Scientific)
  • 22 × 44–mm coverslips
  • ASA 100 color film

Basic Protocol 3: Evaluation of Chromosome Aberrations by Painting with Fluorescence In Situ Hybridization (FISH) Probes

  Materials
  • Chromosome‐specific DNA painting probes (2 chromosomes) for the species of interest, labeled with biotin or digoxigenin (Vysis, Inc.)
  • Hybridization solution (see recipe)
  • Metaphase spreads (see protocol 4) from test‐agent‐treated cells
  • 70% (v/v) formamide/2× SSC and 50% (v/v) formamide/2× SSC (see recipe for SSC)
  • 70%, 80%, and 100% ethanol
  • Rubber cement
  • 2× SSC (see recipe)/1% (v/v) NP‐40 (Ventana Medical Systems)
  • Sodium phosphate (PN) buffer (see recipe)
  • 3% (w/v) powdered non‐fat milk in recipePN buffer
  • 5 µg/ml FITC‐avidin in PN buffer
  • 12 µg/ml biotinylated anti‐avidin in PN buffer (optional)
  • 200 µg/ml stock DAPI stain (store up to 1 year at −20°C; dilute to 0.5 µg/ml in antifade solution just before use)
  • 200 µg/ml stock propidium iodide stain (store up to 1 year at −20°C; dilute to 1 µg/ml in antifade solution just before use)
  • Antifade solution (Vysis, Inc., Molecular Probes, Ventana Medical Systems)
  • Coplin jars
  • Humidified chamber (container with lid and lined with moist paper towels)
  • 22‐mm2 coverslips
  • Kodak Ektachrome 400 film

Support Protocol 1: Preparation of Metaphase Chromosome Spreads

  Materials
  • Cells to be studied
  • Medium and supplements appropriate for the cells being studied
  • Agent to be tested in appropriate solvent
  • 10 µg/ml colchicine or Colcemid
  • Hypotonic solution: 0.075 M KCl, 37°C
  • Carnoy's fixative: 3:1 (v/v) methanol/glacial acetic acid, freshly prepared (ice‐cold)
  • 60‐mm petri dishes
  • Microscope slides, cleaned and chilled

Basic Protocol 4: Detection of Sister Chromatid Exchanges

  Materials
  • Cells to be studied
  • Medium and supplements appropriate for the cell line studied ( appendix 3B)
  • Agent to be tested
  • 0.5 mM 5‐bromodeoxyuridine (BrdU), filter sterilized, (store up to 2 to 3 weeks in the dark at −20°C)
  • 10 µg/ml colchicine or Colcemid
  • Hypotonic solution: 0.075 M KCl
  • Carnoy's fixative 3:1 (v/v) methanol/glacial acetic acid (freshly prepared), ice‐cold
  • 2 mg/ml Hoechst 33258 in water (store several months in the dark at −20°C)
  • 0.5× and 2× SSC (see recipe)
  • 3% (v/v) Giemsa solution (prepare fresh): dilute Giemsa stain (see recipe) 3:97 (v/v) in Sorenson's phosphate buffer, pH 6.8 (see recipe)
  • 60‐ and 100‐mm petri dishes
  • Microscope slides, cleaned and chilled
  • UV light source (Philips MWL 160 W lamp)
  • Additional reagents and equipment for mutagenesis of cells (unit 3.3), trypsinization of cultures ( appendix 3B), and preparation of metaphase spreads (see protocol 4)
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Figures

Videos

Literature Cited

Literature Cited
   Bangs, C.D. and Donlon, T.A. 1994. Chromosome preparation from peripheral blood cells. In Current Protocols in Human Genetics N.C. Dracopoli, J.L. Haines, B.R. Korf, D.T. Moir, C.C. Morton, C.E. Seidman, J.G. Seidman and D.R. Smith, Eds., pp. 4.1.1‐4.1.19 John Wiley & Sons, New York.
   Boei, J.J.W.A., Vermeulen, S., and Natarajan, A.T. 1998. Dose‐response curves for X‐ray‐induced interchanges and interarm interchanges in human lymphocytes using arm‐specific probes for chromosome 1. Mutat. Res. 404:45‐53.
   Christian, A.T., Garcia, H.E., and Tucker, J.D. 1999. PCR in situ followed by microdissection allows whole chromosome painting probes to be made from single microdissected chromosomes. Mammalian Genome. 10(6):628‐631.
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   Grigorova, M. and Natarajan, A.T. 1998. Relative involvement of chromosome #21 in radiation induced exchange aberrations in lymphocytes of Down syndrome patients. Mutat. Res. 404:67‐75.
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   Salassidis, K., Huber, R., Zitzelberger, H., and Bauchinger, M. 1992. Centromere detection in vinblastine‐ and radiation‐induced micronuclei of cytokinesis‐blocked mouse cells by using in situ hybridization with a mouse gamma (Major) satellite DNA probe. Environ. Mol. Mutagen. 19:1‐6.
   Schröck, E., du Manoir, S., Veldman, T., Schoell, B., Wienberg, J., FergusonSmith, M.A., Ning, Y., Ledbetter, D.H., Bar‐Am, I., Soenksen, D., Garini, Y., and Ried, T. 1996. Multicolor spectral karyotyping of human chromosomes. Science 273:494‐497.
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   Tucker, J.D., Auletta, A., Cimino, M.C., Dearfield, K.L., Jacobson, Kram D., Tice, R.R., and Carrano, A.V. 1993. Sister‐chromatid exchange: Second report of the Gene‐Tox program. Mutat. Res. 297:101‐180.
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