Construction of Somatic Cell Hybrids

Cynthia L. Jackson1

1 Rhode Island Hospital and Brown University, Providence, Rhode Island
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 3.2
DOI:  10.1002/0471142905.hg0302s09
Online Posting Date:  May, 2001
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Abstract

Somatic cell hybridization is the method of choice to separate a chromosome of interest from the full chromosome complement and obtain a permanent source of the chromosome. This unit begins with the choice of fusion techniques and selectable markers for hybrids containing a chromosome of interest. The first set of protocols outline the production of whole‐cell hybrids by fusion of two cell lines: a monolayer (adherent) recipient and a donor that may be adherent or grown in suspension. The second set of protocols outline the production of micronuclei containing a limited number of chromosomes, and enucleation of the micronuclei to form microcells for fusion with recipient cells. Support protocols describe the preparation and use of cloning cylinders to isolate colonies in tissue culture, subcloning of whole‐cell hybrid populations to isolate lines that have segregated additional chromosomes, purification of microcell preparations, and molecular and cytogenetic methods for characterizing.

     
 
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Table of Contents

  • Strategic Planning
  • Selectable Markers for Mammalian Cells
  • Basic Protocol 1: Whole‐Cell Fusion of Monolayer Cells
  • Alternate Protocol 1: Whole‐Cell Fusion of Suspension Donor Cells to Monolayer Recipient Cells
  • Support Protocol 1: Colony Isolation Using Cloning Cylinders
  • Support Protocol 2: Subcloning Hybrid Cell Populations
  • Basic Protocol 2: Microcell Fusion of Monolayer Cells Using Untreated Plastic Bullets and Cytochalasin B
  • Alternate Protocol 2: Microcell Fusion of Monolayer Cells Using Plastic Bullets Treated with Concanavalin A
  • Alternate Protocol 3: Microcell Fusion of Suspension Donor Cells to Monolayer Recipient Cells
  • Support Protocol 3: Purification of Microcell Preparations
  • Characterization of Hybrids
  • Support Protocol 4: Cytogenetics: G‐11 Staining of Metaphase Chromosomes
  • Support Protocol 5: In Situ Hybridization (ISH)
  • Support Protocol 6: Marker Analysis
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Whole‐Cell Fusion of Monolayer Cells

  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 .
  • Confluent culture of recipient cells (see and Table 3.2.1)
  • Appropriate selective agent (see )
  • Serum‐containing tissue culture medium appropriate for recipient cells (e.g., complete DMEM/10% FBS, appendix 3G), prewarmed to 37°C
  • Corresponding serum‐free medium, 37°C
  • Donor cells containing chromosomes of interest and appropriate genetic markers (see )
  • 50% (w/v) PEG 1000 solution (see recipe), 37°C
  • 100‐mm tissue culture plates
  • Inverted phase‐contrast microscope
  • 6‐ or 24‐well tissue culture plates
  • Additional reagents and equipment for culturing and trypsinizing cells ( appendix 3G)
CAUTION: Human donor cells are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 1: Whole‐Cell Fusion of Suspension Donor Cells to Monolayer Recipient Cells

  Additional 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 cell line growing in suspension (see ) or fresh lymphocytes from whole blood ( appendix 3B)
  • Serum‐free medium, prewarmed to 37°C
  • 15‐ml snap‐cap polypropylene tubes
  • IEC Clinical centrifuge
  • Additional reagents and equipment for counting cells ( appendix 3G)
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 1: Colony Isolation Using Cloning Cylinders

  Additional 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.
  • Tissue culture cells on plate
  • PBS ( appendix 2D), 37°C
  • Sterile vacuum grease (place in glass petri dish and sterilize by autoclaving)
  • Trypsin/EDTA solution: 5% (w/v) trypsin/2% (w/v) EDTA (store in aliquots at −20°C), prewarmed to 37°C
  • Cloning cylinders (see recipe)
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 2: Subcloning Hybrid Cell Populations

  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.
  • Cloned hybrid cell line ( protocol 1basic or protocol 2alternate protocols)
  • Serum‐containing tissue culture medium appropriate for hybrid cell line (recipient cells): e.g., complete DMEM/10% FBS ( appendix 3G)
  • 96‐well microtiter plate
  • Additional reagents and equipment for counting cells ( appendix 3G)
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: Microcell Fusion of Monolayer Cells Using Untreated Plastic Bullets and Cytochalasin B

  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.
  • Selective agent (see )
  • Recipient cells (see and Table 3.2.1)
  • Serum‐containing tissue culture medium appropriate for recipient cell line (e.g., complete DMEM/10% FBS, appendix 3G), 37°C and room temperature
  • Corresponding serum‐free medium, 37°C and room temperature
  • Monolayer culture of donor cells containing chromosome of interest and appropriate genetic markers (see )
  • Serum‐containing tissue culture medium ( appendix 3G) appropriate for donor cell line, 37°C
  • 1 mg/ml Colcemid solution (see recipe)
  • 2 mg/ml cytochalasin B (Aldrich) in DMSO (store ≤6 months at 4°C)
  • 0.5% (w/v) SDS
  • Aceto‐orcein solution (see recipe)
  • 100 µg/ml PHA‐P in appropriate medium (see recipe)
  • 44% (w/v) PEG 1000 solution (see recipe), 37°C
  • 150‐mm tissue culture plates
  • Plastic bullets (see recipe)
  • 50°C oven
  • Inverted and noninverted phase‐contrast microscopes
  • Beckman J‐2 centrifuge with JA‐20 rotor or Sorvall RC‐5 centrifuge with SS‐34 rotor (or equivalent)
  • 50‐ml round‐bottom polycarbonate centrifuge tubes with caps (Nalgene), sterile
  • 25‐cm2 tissue culture flasks
  • Tissue culture chamber slides
  • Additional reagents and equipment for tissue culture, cell counting, and trypsinization ( appendix 3G), Giemsa staining (unit 8.7), and chromosome staining (unit 4.2)
CAUTION: Human donor cells are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 2: Microcell Fusion of Monolayer Cells Using Plastic Bullets Treated with Concanavalin A

  Additional 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.
  • 75 mg/ml 1‐cyclohexyl‐3‐(2‐morpholinoethyl)‐carbodiimide metho‐p‐toluenesulfonate in PBS (e.g., Sigma; prepare fresh and filter sterilize)
  • Concanavalin A (Con A) solution (see recipe)
  • PBS ( appendix 2D)
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 3: Microcell Fusion of Suspension Donor Cells to Monolayer Recipient Cells

  Additional 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: suspension culture of lymphoblast cells (see )
  • Percoll (Pharmacia Diagnostics)
  • 100 µg/ml PHA‐P in HBSS (see recipe)
CAUTION: Human donor cells are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 3: Purification of Microcell Preparations

  Additional 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.
  • Microcell preparation (step of second basic protocol or step of second alternate protocol)
  • 10‐ml syringes with Swinnex Luer‐Lok filter adaptors (Millipore)
  • Polycarbonate filters with 8‐, 5‐, and 3‐µm pore sizes and 25‐mm diameter (Costar)
NOTE: All incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Support Protocol 4: Cytogenetics: G‐11 Staining of Metaphase Chromosomes

  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.
  • Cells of human‐rodent hybrid lines to be tested (basic and alternate protocols)
  • Cells of human donor and rodent recipient lines used to construct hybrids
  • Giemsa stain (see recipe)
  • Hydrion buffer: one Hydrion buffer capsule, pH 11.00 (Micro Essential Laboratory) in 100 ml H 2O
  • 100‐ml staining tray
  • 60°C water bath
  • Bright‐field microscope equipped with 10× lens for scanning and 100× oil immersion lens for analysis
  • Additional reagents and equipment for preparation of air‐dried metaphase spreads (unit 4.1)
CAUTION: Human donor cells are hazardous; see appendix 2A for guidelines on handling, storage, and disposal.
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Figures

Videos

Literature Cited

Literature Cited
   Abbott, C. and Povey, S. 1991. Development of human chromosome‐specific PCR primers for the characterization of somatic cell hybrids. Genomics 9:73‐77.
   Athwal, R.S., Smarsh, M., Searle, B.M., and Deo, S.S. 1985. Integration of a dominant selectable marker into human chromosomes and transfer of marked chromosomes to mouse cells by microcell fusion. Somatic Cell Mol. Genet. 11:177‐187.
   Barski, G., Sorieul, S., and Cornefert, F. 1961. Hybrid type cells in combined cultures of two different mammalian cell strains. J. Nat. Cancer Inst. 26:1269‐1291.
   Boni, L.T. and Hui, S.W. 1987. The mechanism of polyethylene glycol induced fusion in model membranes. In Cell Fusion (A.E. Sowers, ed.) pp. 301‐330. Plenum, New York.
   Crenshaw, A.H. and Murrell, L.R. 1982. Monolayer enucleation of colcemid‐treated human cells and polyethylene glycol 400‐mediated fusion of microkaryoplasts (microcells) with whole cells. In Techniques in Somatic Cell Genetics (J.W. Shay, ed.) pp. 291‐305. Plenum, New York.
   Davidson, R.L. and Gerald, P.S. 1976. Improved techniques for the induction of mammalian cell hybridization by polyethylene glycol. Somatic Cell Genet. 2:165‐176.
   Dubois, B.L. and Naylor, S.L. 1993. Characterization of NIGMS human/rodent somatic cell hybrid mapping panel 2 by PCR. Genomics 16:315‐319.
   Ege, T. and Ringertz, N.R. 1974. Preparation of microcells by enucleation of micronucleate cells. Exp. Cell Res. 87:378‐382.
   Fournier, R.E.K. 1981. A general high‐efficiency procedure for production of microcell hybrids. Proc. Natl. Acad. Sci. U.S.A. 78:6349‐6353.
   Fournier, R.E.K. and Moran, R.G. 1983. Complementation mapping in microcell hybrids: Localization of Fpgs and Ak‐1 on Mus musculus chromosome 2. Somatic Cell Genet. 1:69‐84.
   Fournier, R.E.K. and Ruddle, F.H. 1977. Microcell‐mediated transfer of murine chromosomes into mouse, Chinese hamster, and human somatic cells. Proc. Natl. Acad. Sci. U.S.A. 74:319‐323.
   Gillin, F.D., Roufa, D.J., Beaudet, A.L., and Caskey, C.T. 1972. 8‐azaguanine resistance in mammalian cells. I. Hypoxanthine‐guanine phosphoribosyltransferase. Genetics 72:239‐252.
   Kao, F.‐T., Jones, C., and Puck, T.T. 1976. Genetics of somatic mammalian cells: Genetic, immunologic, and biochemical analysis with Chinese hamster cell hybrids containing selected human chromosomes. Proc. Natl. Acad. Sci. U.S.A. 73:193‐197.
   Kurdi‐Haidar, B., Levine, F., Roemer, K., LaPorte, P., and Friedmann, T. 1993. Provirus‐anchored long range mapping of mammalian genomes. Genomics 15:305‐310.
   Leach, R.J., Thayer, M.J., Schafer, A.J., and Fournier, R.E.K. 1989. Physical mapping of human chromosome 17 using fragment‐containing microcell hybrids. Genomics 5:167‐176.
   Ledbetter, S.A., Garcia‐Heras, J., and Ledbetter, D.H. 1990. “PCR‐karyotype” of human chromosomes in somatic cell hybrids. Genomics 8:614‐622.
   Littlefield, J.W. 1964. Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants. Science 145:709‐710.
   Lugo, T.G., Handelin, B., Killary, A.M., Housman, D.E., and Fournier, R.E.K. 1987. Isolation of microcell hybrid clones containing retroviral vector insertions into specific human chromosomes. Mol. Cell. Biol. 7:2814‐2820.
   Mercer, W.E. and Baserga, R. 1982. Techniques for decreasing the toxicity of polyethylene glycol. In Techniques in Somatic Cell Genetics (J.W. Shay, ed.) pp. 23‐33. Plenum, New York.
   Pontecorvo, G. 1975. Production of mammalian somatic cell hybrids by means of polyethylene glycol treatment. Somatic Cell Genet. 1:397‐400.
   Ringertz, N.R. and Savage, R.E. 1976. Cell Hybrids. Academic Press, San Diego.
   Sanford, J.A. and Stubblefield, E. 1987. General protocol for microcell‐mediated chromosome transfer. Somatic Cell Mol. Genet. 13:279‐284.
   Saxon, P.J., Srivatsan, E.S., Leipzig, G.V., Sameshima, J.H., and Stanbridge, E.J. 1985. Selective transfer of individual human chromosomes to recipient cells. Mol. Cell. Biol. 5:140‐146.
   Theune, S., Fung, J., Todd, S., Sakaguchi, A.Y., and Naylor, S.L. 1991. PCR primers for human chromosomes: Reagents for the rapid analysis of somatic cell hybrids. Genomics 9:511‐516.
   Warburton, D., Gersen, S., Yu, M.‐T., Jackson, C., Handelin, B., and Housman, D. 1990. Monochromosomal rodent‐human hybrids from microcell fusion of human lymphoblastoid cells containing an inserted dominant selectable marker. Genomics 6:358‐366.
   Weiss, M.C. and Green, H. 1967. Human‐mouse hybrid cell lines containing partial complements of human chromosomes and functioning human genes. Proc. Natl. Acad. Sci. U.S.A. 58:1104‐1111.
   Weissman, B.E., Saxon, P.J., Pasquale, S.R., Jones, G.R., Geiser, A.G., and Stanbridge, E.J. 1987. Introduction of a normal human chromosome 11 into a Wilms tumor cell line controls its tumorigenic expression. Science 236:175‐180.
Key References
   Shay, J.W., ed. 1982. Techniques in Somatic Cell Genetics. Plenum, New York.
  Contains chapters on all relevant parameters for making hybrids.
   Adolph, K.W., ed 1993. Methods in Molecular Genetics, Vol. 1: Gene and Chromosome Analysis, Part A. Academic Press, San Diego.
  Chapters 6 and 7 contain a good discussion of preparation and application of microcell‐mediated chromosome transfer.
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