Protein Expression in the Drosophila Schneider 2 Cell System

John A. Schetz1, Eswar P.N. Shankar1

1 University of North Texas Health Science Center, Fort Worth, Texas
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 4.16
DOI:  10.1002/0471142301.ns0416s27
Online Posting Date:  September, 2004
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Abstract

The Schneider‐2 (S2) Drosophila cell line is well suited for the stable overexpression of recombinant proteins using plasmid‐based protein expression vectors. Following drug selection, a polyclonal S2 cell line can be induced to express on the order of 2 to 100 pmol/mg membrane protein for G‐coupled protein receptors, 4000 to 100,000 sites/cell for other membrane receptors and 3 to 35 mg/liter for soluble and secreted proteins.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Stable Polyclonal S2 Cell Lines
  • Basic Protocol 2: Protein Expression by Induction of Exogenous Target Genes in S2 Cell Lines
  • Support Protocol 1: Culture and Storage of Drosophila S2 Cells
  • Support Protocol 2: Preparation of a Modified Shields and Sang Complete M3 Medium
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Stable Polyclonal S2 Cell Lines

  Materials
  • Schneider 2 (S2) at or near log phase cells (see step or of protocol 3)
  • Complete M3 medium (see protocol 4)
  • Plasmid DNA purified such that OD 260/280 ≥ 1.8
  • N,N‐Bis(2‐hydroxyethyl)‐2‐aminoethanesulfonic acid (BES) buffer–based calcium phosphate transformation reagents (e.g., Stratagene)
  • 70% (v/v) ethanol
  • Incomplete S2 medium (e.g., Life Technologies)
  • 100× solution of selection drug (optional): e.g., 100 mg/ml geneticin (G418) or 20 to 30 mg/ml hygromycin B
  • 150‐cm2 polystyrene tissue culture dishes
  • 17 × 100–mm 14‐ml tubes (Falcon #2059)
  • 50‐ml conical polypropylene centrifuge tubes with caps
  • Cell lifter
  • 75‐cm2 screw‐cap (nonaerating) polystyrene tissue culture flasks
  • 27°C incubator
  • Additional reagents and equipment for culturing S2 cells (see protocol 3) and testing expression (see protocol 2)

Basic Protocol 2: Protein Expression by Induction of Exogenous Target Genes in S2 Cell Lines

  Materials
  • S2 cultures transfected with gene of interest (see protocol 1)
  • Complete M3 medium (see protocol 4; for Mtn induction)
  • 50 mM CuSO 4or 1 mM CdCl 2 (for Mtn induction): ultrapure (e.g., Aldrich) and sterilized with a 0.22‐µm filter
  • Aluminum foil (for hsp70 induction)
  • 37°C incubator

Support Protocol 1: Culture and Storage of Drosophila S2 Cells

  Materials
  • Schneider 2 (S2) cells (ATCC #CRL‐1963)
  • Complete M3 medium (see protocol 4)
  • Cell culture–grade dimethyl sulfoxide (DMSO)
  • Incomplete S2 medium (e.g., Life Technologies)
  • 25‐ and 150‐cm2 screw‐cap (nonaerating) polystyrene tissue culture flasks
  • 27°C incubator (or other dark, dry environment)
  • 1.8‐ml polypropylene cryotubes
  • Liquid nitrogen storage tank
  • 15‐ml conical polypropylene centrifuge tubes with caps
  • 10‐ml pipet
  • Additional reagents and equipment for counting cells with a hemacytometer ( appendix 3B)

Support Protocol 2: Preparation of a Modified Shields and Sang Complete M3 Medium

  Materials
  • Tissue culture–tested fetal bovine serum (FBS)
  • Powdered modified Shields and Sang M3 insect medium (e.g., Sigma)
  • Tissue culture–grade potassium bicarbonate (KHCO 3)
  • Penicillin/streptomycin at 5000 U/ml and 5 mg/ml, respectively
  • Clear resealable plastic bag (e.g., Ziploc)
  • 65°C circulating water bath
  • 50‐ml polypropylene centrifuge tubes
  • 250‐ml, 0.45‐µm tissue culture–grade sterile filtration devices with glass fiber prefilters
  • 1‐liter, 0.22‐µm tissue culture–grade sterile filtration device with glass fiber prefilter
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Literature Cited

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Key References
   Angelichio et al., 1991. See above.
  The single most extensive comparison of the effectiveness of promoters and polyadenylation signals in S2 cells published to date. A must read for those attempting to design S2 cell expression vectors.
   Ashburner, 1989. See above.
  A comprehensive treatise on Drosophila laboratory techniques, including specific Drosophila cell culture techniques such as cloning procedures for S2 cells. Contains many detailed methodological tips referenced as personal communications with prominent Drosophila researchers.
   Ashburner, M. 1989. Drosophila: A Laboratory Handbook. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  Companion handbook for the manual listed above. In addition to being a good general resource on Drosophila, this book provides historical and factual details about vectors designed for use in Drosophila cells.
   Drosophila Information Services (DIS).
  A series of documents produced yearly from materials contributed by Drosophila researchers. Specific arrangements and bibliographies for this documentation service, as well as the institutions at which these publications are produced, vary according to volume. In most libraries, the DIS is listed as a journal by volume and year. The DIS is a compilation of short blurbs concerning recent methodological advances in Drosophila research, especially those related to Drosophila tissue culture.
Internet Resources
   http://flybase.bio.indiana.edu
  An extensive database resource for all that is Drosophila, including sequence information for various Drosophila vectors (transposons).
   http://www.invitrogen.com/expressions/index.htmlis
  Invitrogen's Web site, containing schematics of their Drosophila expression vectors.
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