Construction and Assay of Radiation Hybrids

Charles W. Richard1, Sarah S. Washington1

1 University of Pittsburgh, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 3.3
DOI:  10.1002/0471142905.hg0303s00
Online Posting Date:  May, 2001
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Abstract

This unit describes the irradiation and fusion of cells to produce radiation hybrids and the harvesting and expansion of RH colonies. A support protocol describes amplification of human chromosome fragments by PCR and analysis of those fragments by agarose gel electrophoresis.

     
 
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Table of Contents

  • Basic Protocol 1: Construction of Radiation Hybrids
  • Support Protocol 1: PCR Analysis of Marker Loci in Radiation Hybrids
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Construction of Radiation Hybrids

  MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • Donor cells: somatic HPRT+ human‐hamster hybrid cells containing an intact human chromosome as the only human material (units 3.1 & 3.2)
  • Complete tissue culture media appropriate for donor and recipient cells ( appendix 3G; e.g., complete DMEM, Ham F‐12, or RPMI) with serum, 37°C and 4°C
  • Tissue culture media appropriate for donor and recipient cells without serum, HAT, penicillin, or streptomycin (unsupplemented medium), 37°C and 4°C
  • Recipient cells: somatic HPRT cell line, preferably of same background as donor cells (e.g., 380‐6, GM00459, UCW113, CHTG49, or A9, available from ATCC or Coriell Cell Repositories)
  • 8‐azaguanine, 10× (GIBCO/BRL) or 50× (Sigma)
  • Trypsin/EDTA solution ( appendix 3G), 37°C
  • HAT medium: 50× HAT supplement (Sigma) diluted 1:49 in complete medium with serum appropriate for recipient cells, 37°C
  • 50% (w/v) PEG 1500 (Boehringer Mannheim), 37°C
  • IEC Clinical centrifuge (or equivalent) with swinging‐bucket rotor
  • 60Co or137Cs γ‐irradiator (e.g., Gammacell 1000, Nordion International)
  • 10 × 75–mm conical snap‐cap polypropylene tubes, sterile
  • Inverted microscope
  • 24‐well tissue culture plates
  • Additional reagents and equipment for tissue culture, cell counting, trypsinization, and freezing of tissue culture cells ( appendix 3G), isolating clones using cloning cylinders (unit 3.2), and isolating DNA ( appendix 3B)
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: PCR Analysis of Marker Loci in Radiation Hybrids

  MaterialsFor recipes, see Reagents and Solutions in this unit (or cross‐referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • DNA solutions prepared from 94 radiation‐hybrid colonies plus donor and recipient cell lines ( protocol 1basic protocol)
  • PCR primers selective for human marker sequences
  • Tartrazine dye (Aldrich) diluted to 1 mg/ml in H 2O
  • Mineral oil
  • PCR reaction mix (see recipe)
  • 96‐well microtiter plate (e.g., flexible polycarbonate plates, Techne)
  • Biomek 1000 robotic workstation with multipipetting tool (Beckman; optional)
  • 8‐ or 12‐channel pipettor and tips
  • Plate sealer tape (e.g., Costar)
  • Thermal cycler (e.g., PHC‐3, Techne)
  • Custom gel electrophoresis comb with spacing for microtiter‐well format
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)
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Figures

Videos

Literature Cited

Literature Cited
   Cox, D.R., Burmeister, M., Price, E.R., Kim, S., and Myers, R.M. 1990. Radiation hybrid mapping: A somatic cell genetic method for constructing high resolution maps of mammalian chromosomes. Science 250:245‐250.
   Douglas, W.H.J. and Dell'Orco, R.T. 1979. Physical aspects of a tissue culture laboratory. Methods Enzymol. 58:3‐18.
  Généthon Human Genome Research Centre, 1992. The Généthon microsatellite map catalogue. Généthon Human Genome Research Centre, Evry, France.
   Goss, S.J. and Harris, H. 1975. New methods for mapping genes in human chromosomes. Nature 255:680‐684.
   Ham, R.G. and McKeehan, W. L. 1979. Media and growth requirements. Methods Enzymol. 58:44‐93.
   Kennett, R.G. 1979. Cell fusion. Methods Enzymol. 58:345‐359.
Key Reference
   Cox et al., 1990 See above.
  Original paper describing the refinement of RH mapping for making chromosomal maps.
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