Transfection of Lymphoid Cells

Sherie L. Morrison1

1 University of California Los Angeles, Los Angeles, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.17B
DOI:  10.1002/0471142735.im1017bs31
Online Posting Date:  May, 2001
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Abstract

Using available vectors, a single immunoglobulin chain or both heavy (H) and light (L) chains can be simultaneously transferred to recipient myeloma cells. Several methods can be used to prepare stable transfected lymphoid cells. The in this unit describes protoplast fusion, which is suitable for certain lymphoid cells but tends to be more cumbersome than electroporation or liposomeā€mediated transfection, which are described in the two alternate protocols. All three techniques may be adapted for stable transfection of other eukaryotic cell lines.

     
 
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Table of Contents

  • Basic Protocol 1: Protoplast Fusion for Transfection of Lymphoid Cells
  • Alternate Protocol 1: Electroporation for Stable Transfection of Lymphoid Cells
  • Alternate Protocol 2: Liposome‐Mediated Stable Transfection of Nonadherent Lymphoid Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Protoplast Fusion for Transfection of Lymphoid Cells

  Materials
  • E. coli HB101 containing immunoglobulin expression plasmid (unit 2.12)
  • LB medium (unit 10.18) containing appropriate antibiotic
  • 20 mg/ml ampicillin
  • 30 mg/ml chloramphenicol in ethanol (filter sterilize)
  • LB medium containing 1% (w/v) glucose
  • 10× chloramphenicol: 1.25 mg/ml in LB medium (filter sterilize and store at 4°C)
  • 10× spectinomycin: 1.5 mg/ml in LB medium (filter sterilize and store at 4°C)
  • 20% (w/v) sucrose/50 mM Tris⋅Cl, pH 8.0 (filter sterilize), ice‐cold
  • 5 mg/ml lysozyme/250 mM Tris⋅Cl, pH 8.0 (filter sterilize)
  • 250 mM EDTA, pH 8.0, ice‐cold
  • 50 mM Tris⋅Cl, pH 8.0, ice‐cold
  • Dulbeccos modified Eagle medium (DMEM; appendix 2A) containing 20% (w/v) sucrose and 10 mM MgCl 2
  • Recipient cells: 2–5 × 106 exponentially growing myeloma cells (e.g., J558L, Sp2/0, or PBX63Ag8653, ATCC)
  • Complete Dulbeccos modified Eagle medium (DMEM), room temperature and 37°C
  • PEG/DMSO solution: 41.7% (w/v) polyethylene glycol (PEG) 1500/12.5% (v/v) DMSO in 100 mM Tris⋅Cl, pH 8.0, 37°C
  • PEG/Tris/DMEM solution: 50% (w/v) polyethylene glycol (PEG) 1500/100 mM Tris⋅Cl, pH 8.0/DMEM, 37°C
  • Complete DMEM‐10 ( appendix 2A) containing 100 µg/ml gentamicin and 100 U/ml nystatin, 37°C
  • 250‐ml centrifuge bottle with screw‐cap, sterile
  • Sorvall centrifuge with GSA rotor for 250‐ml bottles
  • 50‐ml polypropylene tubes, sterile
  • IEC centrifuge with a swinging‐bucket rotor for 50‐ml tubes (or equivalent)
  • 96‐well flat‐bottom microtiter plates
  • 24‐well tissue culture plates
  • Additional reagents and equipment for growing bacterial cells (unit 10.3), cloning by limiting dilution (unit 2.5), freezing cell lines ( appendix 3G), and characterizing immunoglobulin production by transfectants (unit 2.12)
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 1: Electroporation for Stable Transfection of Lymphoid Cells

  • DNA from expression plasmid bearing immunoglobulin heavy (H) and/or light (L) chain (units 2.12 & 10.3)
  • Appropriate restriction endonuclease and buffer for linearization of plasmid (unit 10.8; optional)
  • Phosphate‐buffered saline (PBS) without Mg2+ and Ca2+ ( appendix 2A), sterile
  • Complete Iscoves modified Dulbeccos medium (IMDM, GIBCO/BRL) containing 100 µg/ml gentamicin and 100 U/ml nystatin
  • Electroporation cuvettes
  • Electroporator (Bio‐Rad Gene Pulser or equivalent)
  • Additional reagents and equipment for phenol extraction and ethanol precipitation of DNA (unit 10.1) and preparation of recipient cells for electroporation (unit 10.15)
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Alternate Protocol 2: Liposome‐Mediated Stable Transfection of Nonadherent Lymphoid Cells

  • Exponentially growing recipient cells—e.g., nonadherent lymphoid cells
  • Purified DNA from expression plasmid bearing immunoglobulin heavy (H) and/or light (L) chain (units 2.12 & 10.3)
  • Liposome suspension (Lipofectin, GIBCO/BRL)
  • Serum‐free Iscoves modified Dulbeccos medium (IMDM)
  • recipeSupplemented Iscoves modified Dulbeccos medium (IMDM, see recipe)
  • 15‐ml polystyrene tube
  • 60‐mm plastic petri dish
NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.
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Figures

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Literature Cited

Literature Cited
   Felgner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P., Ringold, G.M., and Danielson, M. 1987. Lipofectin: A high efficient, lipid‐mediated DNA/transfection procedure. Proc. Natl. Acad. Sci. U.S.A. 84:7413‐7417.
   Oi, V.T. and Morrison, S.L. 1986. Chimeric antibodies. BioTechniques 4:214‐221.
   Potter, H., Weir, L., and Leder, P. 1984. Enhancer‐dependent expression of human κ immunoglobulin genes introduced into mouse pre‐B lymphocytes by electroporation. Proc. Natl. Acad. Sci. U.S.A. 81:7161‐7165.
   Sandri‐Goldin, R.M., Goldin, A.L., Levine, M., and Glorioso, J.C. 1981. High‐frequency transfer of clonal herpes simplex virus type I sequences to mammalian cells by protoplast fusion. Molec. Cell Biol. 1:743‐752.
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