Use of Monoclonal Antibodies for Expression Cloning

Diane Hollenbaugh1, Alejandro Aruffo1, Bryan Jones1, Peter Linsley1

1 Bristol‐Myers Squibb, Seattle, Washington
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.18
DOI:  10.1002/0471142735.im1018s31
Online Posting Date:  May, 2001
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Abstract

This unit details the use of transient expression in mammalian cells to screen cDNA libraries with monoclonal antibodies (MAb) to isolate cDNA clones encoding cell‐surface and intracellular proteins. The first protocol describes the cloning of cDNAs encoding cell‐surface antigens. The second protocol is a modification that facilitates isolation of cDNAs encoding antigens that are expressed intracellularly. Both protocols are designed for use with the expression vector CDM8, which contains a polylinker for subcloning double‐stranded cDNA.

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of cDNA Clones Encoding Cell‐Surface Antigens
  • Support Protocol 1: Preparation of Antibody‐Coated Dishes
  • Basic Protocol 2: Isolation of cDNA Clones Encoding Intracellular Antigens
  • Support Protocol 2: Preparation of Polyvinylidene‐Wrapped Dishes
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of cDNA Clones Encoding Cell‐Surface Antigens

  Materials
  • Complete Dulbeccos minimum essential medium containing 10% NuSerum or 10% calf serum (complete DMEM‐10 NS or complete DMEM‐10 CS; appendix 2A) or complete Iscoves modified Dulbeccos medium containing 10% NuSerum (complete IMDM‐10 NS; appendix 2A)
  • 100‐mm tissue culture dishes seeded with COS cells (∼50% confluent)
  • Plasmid DNA containing >106 of cDNA clones (CsCl purified; units 10.3 & 10.13; see 10.18background information)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 10 mg/ml DEAE‐dextran + 2.5 mM chloroquine in PBS
  • 10% (v/v) DMSO in PBS
  • 0.5 mM EDTA/0.02% (v/v) azide in PBS
  • 0.5 mM EDTA/0.02% (v/v) azide/5% (v/v) calf serum in PBS
  • 0.5 mg/ml trypsin/0.2 mg/ml EDTA in PBS
  • 1 µg/ml purified monoclonal antibody (MAb) or 1:100 dilution of ascites fluid (unit 2.6)
  • 0.5 mM EDTA/0.02% (v/v) azide/2% (w/v) Ficoll
  • 60‐mm antibody‐coated dishes (first protocol 2support protocol)
  • 5% (v/v) calf serum in PBS
  • 0.6% (w/v) SDS/ 10 mM EDTA
  • 5 M NaCl
  • Phenol (extracted twice with 1 M Tris⋅Cl, pH 7.5)
  • 2 µg/µl linear polyacrylamide
  • 100% ethanol
  • TE buffer, pH 7.5 ( appendix 2A)
  • 3 M sodium acetate ( appendix 2A), pH 5.2 or with pH not adjusted
  • Electroporation‐competent E. coli cells (Seidman and Sheen, )
  • recipeLB medium
  • 100 mg/ml spectinomycin or 35 mg/ml chloramphenicol in ethanol
  • 20% (w/v) sucrose/50 mM Tris⋅Cl, pH 8.0, ice cold
  • 5 mg/ml lysozyme (Sigma #L6876), freshly prepared in 250 mM Tris⋅Cl, pH 8.0
  • 250 mM EDTA, ice cold
  • 50 mM Tris⋅Cl, pH 8.0
  • 10% (w/v) sucrose/GIBCO/BRL #320‐1960AJ) without serum, filter sterilized
  • 60‐mm tissue culture dishes seeded with COS cells (∼50% confluent)
  • 50% (w/w) PEG 1000 or 1450 in DMEM (no serum), adjusted to pH 7 with 7.5% (w/v) sodium bicarbonate (Baker or Kodak)
  • DMEM without serum
  • Complete DMEM‐10 CS ( appendix 2A) containing 15 µg/ml gentamicin sulfate
  • Nylon mesh, 100‐µm pore size (Tetko)
  • Sorvall GSA rotor or equivalent
  • Additional reagents and equipment for transformation of E. coli (Seidman et al., ); phenol extraction and ethanol precipitation (unit 10.1), alkaline lysis miniprep (unit 10.3), and flow cytometry (Chapter 5)

Support Protocol 1: Preparation of Antibody‐Coated Dishes

  Additional Materials
  • Anti‐mouse affinity purified antibody, sheep or goat (e.g., Cappel)
  • 50 mM Tris⋅Cl, pH 9.5
  • 0.15 M NaCl
  • 1 mg/ml BSA in PBS ( appendix 2A)
  • 60‐mm bacteriological dishes (e.g., Falcon #1007) or 100‐mm dishes (e.g., Fisher #8757‐12)

Basic Protocol 2: Isolation of cDNA Clones Encoding Intracellular Antigens

  Materials
  • 100‐mm tissue culture dishes seeded with COS cells
  • Plasmid DNA containing >106 cDNA clones (CsCl purified; units 10.3 & 10.13; see 10.18background information)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Methanol
  • 1% nonfat dry milk in PBS with and without monoclonal antibody (MAb)
  • 1% nonfat dry milk containing 125I‐labeled protein A
  • 0.6% SDS/ 10 mM EDTA buffer
  • Polyvinylidene‐wrapped dishes (second protocol 4support protocol)
  • X‐ray film
  • Polyvinylidene wrap (e.g., Saran Wrap)
  • Rubber cement
  • Luminescent stickers
  • Additional reagents and equipment for alkaline lysis miniprep (unit 10.3); trypsinization (unit 10.2), and autoradiography ( appendix 3J)

Support Protocol 2: Preparation of Polyvinylidene‐Wrapped Dishes

  Additional Materials
  • Chloroform
  • 70% ethanol
  • 0.1 mg/ml poly‐L‐lysine HCl (Sigma) in 50 mM Tris⋅Cl, pH 8.0, freshly prepared
  • 100‐mm or 60‐mm tissue culture dishes
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Figures

Videos

Literature Cited

Literature Cited
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   Aruffo, A. and Seed, B. 1987. Molecular cloning of a CD28 cDNA by a high‐efficiency COS cell expression system. Proc. Natl. Acad. Sci. U.S.A. 84:8753‐8577.
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   Seidman, C.E., Struhl, K., and Sheen, J. 1989. Introduction of plasmid DNA into cells. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 1.8.1‐1.8.8. Greene Publishing and Wiley‐Interscience, New York.
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   Tsai, S.F., Martin, D.I., Zon, L.I., D'Andrea, A.D., Wong, G.G., and Orkin, S.H. 1989. Cloning of cDNA for the major DNA‐binding protein of the erythroid lineage through expression cloning. Nature (Lond.) 339:446‐451.
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Key References
   Aruffo and Seed 1987; Seed and Aruffo, 1987. See above.
  Contains original description of cDNA library construction in CDM8 and isolation of cDNA clones encoding cell‐surface antigens by expression cloning.
   Metzelaar et al., 1991; Munro and Maniatis, 1989. See above.
  Contains description of growth of COS cells on wrap and screening for extracellular ligands.
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