Mitotic Chromosome Preparations from Mouse Cells for Karyotyping

Ellen C. Akeson1, Muriel T. Davisson1

1 The Jackson Laboratory, Bar Harbor, Maine
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 4.10
DOI:  10.1002/0471142905.hg0410s25
Online Posting Date:  May, 2001
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Abstract

This unit contains protocols for the preparation of mitotic chromosomes from mouse peripheral blood, Giemsa banding of those chromosomes, and classification into a karyotype, including recognition of some common pitfalls of misidentification and information for determining aberrant chromosomes. The methods described can be used to identify visible chromosomal rearrangements and their precise cytological breakpoints in the living mouse. In conjunction with fluorescent in situ hybridization (FISH), the metaphase spreads can also be used for the linear placement of loci on a chromosome and for determining the insertion site(s) of a foreign transgene.

     
 
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Table of Contents

  • Basic Protocol 1: Culture and Metaphase Harvest of Mouse Peripheral Blood
  • Alternate Protocol 1: Collecting Blood by Tail Vein Method
  • Alternate Protocol 2: Chromosome Preparation from Fetal Liver
  • Support Protocol 1: Setting Up Timed Mouse Matings
  • Alternate Protocol 3: Chromosome Preparation from Bone Marrow
  • Alternate Protocol 4: Chromosome Preparation from Solid Adult Tissues or Tumors
  • Basic Protocol 2: Chromosome Banding
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Culture and Metaphase Harvest of Mouse Peripheral Blood

  Materials
  • recipeComplete RPMI (see recipe)
  • recipePHA solution (see recipe)
  • recipe750 µg/ml LPS solution (see recipe)
  • FBS ( appendix 2D)
  • Female mice, 7 to 10 weeks old
  • 500 USP U/ml recipesodium heparin solution (see recipe)
  • recipe50 µg/ml colchicine solution (see recipe)
  • 0.075 M (0.56% w/v) KCl solution, freshly prepared and prewarmed to 37°C
  • Fixative solution: 3:1 (v/v) methanol/glacial acetic acid, freshly prepared
  • Sterile 16 × 125–mm polystyrene tissue culture tubes with screw cap (Fisher)
  • Heparinized 75‐µl micro‐hematocrit capillary tubes (Fisher)
  • Sterile 12 × 75–mm snap cap tubes (Fisher)
  • 5‐ml conical glass centrifuge tubes (e.g., Kimax, available from Fisher)
  • Clinical benchtop centrifuge (e.g., IEC with 221 rotor), with maximum RCF of 1625 × g
  • Precleaned microscope slides (e.g., Fisher Colorfrost)
  • Phase microscope with 10× or 20× objective for scanning slides; 40× if typing unstained chromosomes
  • Additional reagents and equipment for collecting blood from mouse retro‐orbital sinus (e.g., Donovan and Brown, ) or from mouse tail vein (see protocol 2)

Alternate Protocol 1: Collecting Blood by Tail Vein Method

  Materials
  • Female mouse, 7 to 10 weeks old
  • 70% (v/v) ethanol
  • recipe500 USP U/ml sodium heparin solution (see recipe)
  • Jar
  • Desk lamp
  • Restraining device
  • Razor blade
  • Sterile 12 × 75–mm snap‐cap tube (Fisher)

Alternate Protocol 2: Chromosome Preparation from Fetal Liver

  • Female mouse, pregnant with 14‐day‐old embryos (see protocol 4)
  • recipe0.5% (w/v) colchicine solution (see recipe)
  • recipeEDTA buffer with 0.025% (w/v) colchicine (see recipe; optional)
  • Culture medium, any kind, prewarmed to 37°C
  • Surgical tools for removing embryonic livers
  • 60‐mm Petri dish
  • Small glass dish (∼35‐mm diameter)
  • Dewecker iris scissors
  • 15‐ml conical centrifuge tubes (e.g., Corning)

Support Protocol 1: Setting Up Timed Mouse Matings

  • recipe0.5% (w/v) colchicine solution (see recipe)
  • Surgical tools
  • 1‐ml disposable syringes with 23‐G needles
  • 15‐ml conical centrifuge tubes (Corning)

Alternate Protocol 3: Chromosome Preparation from Bone Marrow

  • Culture medium, any kind, prewarmed to 37°C
  • Surgical tools for removing tumor or tissue
  • 47‐mm Petri dish
  • Small glass dish (∼35 mm diameter)
  • Dewecker iris scissors
  • 15‐ml conical centrifuge tubes (e.g., Corning)

Alternate Protocol 4: Chromosome Preparation from Solid Adult Tissues or Tumors

  Materials
  • Slides of metaphase chromosomes (see protocol 1, or see protocol 2 to protocol 64)
  • 2× SSC ( appendix 2D)
  • 0.9% (w/v) NaCl
  • recipeTrypsin/Giemsa solution (see recipe)
  • Phosphate buffer: 1:1 (v/v) Gurr's phosphate buffer, pH 6.8 (Bio/medical Specialties) in deionized water
  • Coplin jars
  • Bright‐field microscope with 20× objective, 100× oil‐immersion objective, and a green filter
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Figures

Videos

Literature Cited

Literature Cited
   Akeson, E.C. and Davisson, M.T. 1996. Centromeric heterochromatin variants. In Genetic Variants and Strains of the Laboratory Mouse, Vol. 2 (M.F. Lyon, S. Rastan, and S.D.M. Brown, eds.) pp. 1506‐1509. Oxford University Press, Oxford.
   Akeson, E.C. and Davisson, M.T. 2000. Analysing mouse chromosomal rearrangements with G‐banded chromosomes. In Mouse Genetics and Transgenics: A Practical Approach (I. Jackson and C. Abbott, eds.) pp. 144‐153. Oxford University Press, Oxford.
   Andersson, L. and Häyry, P. 1972. Φ‐Isoantigenic marker in phytohemagglutinin responding mouse blood lymphocytes. Experientia 28:81‐82.
   Buckland, R.A., Evans, H.J., and Sumner, A.T. 1971. Identifying mouse chromosomes with the ASG technique. Exp. Cell Res. 69:231‐236.
   Comings, D.E., Avelino, E., Okada, T.A., and Wyandt, H.E. 1973. The mechanism of C‐ and G‐banding of chromosomes. Exp. Cell Res. 77:469‐493.
   Committee on Standardized Genetic Nomenclature for Mice. 1971. Standard karyotype of the mouse, Mus musculus. J. Hered. 63:69‐72.
   Cowell, J.K. 1984. A photographic representation of the variability in the G‐banded structure of the chromosomes in the mouse karyotype. A guide to the identification of the individual chromosomes. Chromosoma (Berl) 89:294‐320.
   Davisson, M.T. and Akeson, E.C. 1987. An improved method for preparing G‐banded chromosomes from mouse peripheral blood. Cytogenet. Cell Genet. 45:70‐74.
   Donovan, J. and Brown, P. 2000. Blood collection. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 1.7.1‐1.7.8. John Wiley & Sons, New York.
   Evans, E.P. 1987. Karyotyping and sexing of gametes, embryos and fetuses and in situ hybridization to chromosomes. In Mammalian Development: A Practical Approach (M. Monk, ed.) pp.93‐114. Oxford University Press, Oxford.
   Evans, E.P. 1996. Standard idiogram. In Genetic Variants and Strains of the Laboratory Mouse, Vol. 2 (M.F. Lyon, S. Rastan, and S.D.M. Brown, eds.) pp. 1446‐1448. Oxford University Press, Oxford.
   Francke, U. and Nesbitt, M. 1971. Identification of the mouse chromosomes by quinacrine mustard staining. Cytogenetics 10:356‐366.
   Han, T. and Pauly, J. 1972. Simplified whole blood method for evaluating in vitro lymphocyte reactivity of laboratory animals. Clin. J. Immunol. 11:137‐142.
   Heiniger, H.J., Taylor, B.A., Hards, E.J., and Meier, H. 1975. Heritability of the phytohemagglutinin responsiveness of lymphocytes and its relation ship to leukemogenesis. Cancer Res. 35:825‐831.
   Hetherington, C.M., Doe, B., and Hay, D. 2000. Mouse care and husbandry. In Mouse Genetics and Transgenics: A Practical Approach (I.J. Jackson and C.M. Abbott, eds.) pp. 17‐18. Oxford University Press, Oxford.
   Hogan, B., Beddington, R., Costantini, F., and Lacy, E. 1994. Maniplating the Mouse Embryo: A Laboratory Manual, 2nd ed. p.129. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Lee, J.L., Warburton, D., and Robertson, E.J. 1990. Cytogenetic methods for the mouse: Preparation of chromosomes, karyotyping, and in situ hybridization. Anal. Biochem. 189:1‐17.
   Nesbitt, M.N. and Francke, U. 1973. A system of nomenclature for band patterns of mouse chromosomes. Chromosoma (Berl) 41:145‐158.
   Nowell, P.C. 1960. Phytohemagglutinin: An initiator of mitosis in cultures of normal human leukocytes. Cancer Res. 20:462‐466.
   Sawyer, J.R., Moore, M.M., and Hozier, J.C. 1987. High resolution G‐banded chromosomes of the mouse. Chromosoma (Berl) 95:350‐358.
   Stobo, J.D., Rosenthal, A.S., and Paul, W.E. 1972. Functional heterogeneity of murine lymphoid cells. I. Responsiveness to and surface binding of concanavalin A and phytohemagglutinin. J. Immunol. 108:1‐17.
   Triman, K.L., Davisson, M.T., and Roderick, T.H. 1975. A method for preparing chromosomes from peripheral blood in the mouse. Cytogenet. Cell Genet. 15:166‐176.
   Wurster, D.H. 1972. Mouse chromosomes identified by trypsin‐Giemsa (T‐G) banding. Cytogenetics 11:379‐387.
Key References
   Cowell, 1984. See above.
  A photographic representation of the G‐banded structure of mouse chromosomes at different stages of condensation with a summary of the main features (landmarks) of each chromosome for identification.
   Evans 1996. See above (pp. 1446‐1450).
  Presents a standard G‐banded karyotype and a pachytene karyotype with an idiogram of the banding patterns of mouse chromosomes after G banding.
   Hogan, B., Beddington, R., Costantini, F., and Lacy, E. 1994. Manipulating the Mouse Embryo: A Laboratory Manual, 2nd ed., pp. 311‐315, 323. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  Procedures for tail bleeding mice, timed matings, preparing embryonic stem cell cultures, preparing slides, and G banding.
   Lee et al., 1990. See above.
  Detailed protocols for preparation of metaphase chromosomes from different sources, with discussions about slide preparation, Giemsa banding, karyotyping, and troubleshooting, and a presentation of a karyogram of mouse strain C57BL/6J and standard idiogram.
   Sawyer et al., 1987. See above.
  A high resolution map of G‐banded mouse chromosomes with a corresponding idiogram.
Internet Resources
   http://www.informatics.jax.org/searches/cytomap.cgi
  Mouse Genome Informatics; Web site for mouse idiogram.
   http://ws4.niai.affrc.go.jp/dbsearch2/mmap/mmap.html
  Animal Genome Database in JAPAN; Web site for mouse idiogram.
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