Inducible Gene Expression Using an Autoregulatory, Tetracycline‐Controlled System

Penny Shockett1, David Schatz2

1 Southeastern Louisiana University, Hammond, Louisiana, 2 Howard Hughes Medical Institute and Yale University School of Medicine, New Haven, Connecticut
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 20.8
DOI:  10.1002/0471143030.cb2008s27
Online Posting Date:  July, 2005
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Abstract

The protocols in this unit describe the transfection of adherent cells and the testing of resultant clones for inducible transactivator or target gene protein expression. Stably transfected fibroblast cell lines expressing transactivator and target gene(s) can be derived by first cotransfecting pTet‐tTAk and a plasmid encoding a selectable marker and obtaining stable lines with inducible transactivator expression. These lines are subsequently stably cotransfected with plasmids encoding the target gene(s) and a second selectable marker. The procedure may also be used to cotransfect pTet‐tTAk with the target gene‐encoding plasmid(s) and a single selectable marker plasmid. A support protocol describes methods to test stably transfected cell lines for inducible gene expression, for transient transfection and induction of tet‐regulated plasmids, and for detection of the tTAk gene in cells (or transgenic mice).

     
 
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Table of Contents

  • Basic Protocol 1: Calcium Phosphate‐Mediated Stable Transfection of NIH3T3 Cells with pTet‐tTAk and Tetracyline‐Regulated Target Plasmids
  • Support Protocol 1: Analysis of Target Gene Protein Expression
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Calcium Phosphate‐Mediated Stable Transfection of NIH3T3 Cells with pTet‐tTAk and Tetracyline‐Regulated Target Plasmids

  Materials
  • NIH3T3 cells
  • Complete DMEM‐10 medium (see recipe)
  • Complete DMEM/tet: complete DMEM‐10 medium (see recipe) containing 0.5 µg/ml tetracycline hydrochloride (Sigma; dilute 10 mg/ml stock in 70% ethanol and store protected from light at −20°C)
  • Selection medium (see recipe) containing 125 µM, 250 µM, or 500 µM L‐histidinol
  • Plasmids for first‐round or cotransfection procedure: pTet‐tTAk (Life Technologies) and plasmids containing target gene ORF(s) cloned into pTet‐Splice (Life Technologies), pSV2‐His, or another selectable marker plasmid; purified by CsCl banding or anion‐exchange chromatography
  • Plasmids for second round transfection procedure: plasmids containing target gene ORF(s) cloned into pTet‐Splice, pPGKPuro, or another selectable marker plasmid; purified by CsCl banding or anion‐exchange chromatography
  • 2 M CaCl 2
  • HEPES‐buffered saline (HeBS; see recipe)
  • 10 mg/ml chloroquine (19 mM; optional; Sigma); dilute in water and store at −20°C
  • 85% (v/v) HeBS/15% (v/v) glycerol, prewarmed to 37°C
  • 3 mg/ml puromycin (Sigma) diluted in PBS ( appendix 2A)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1× trypsin/EDTA (Invitrogen)
  • 10‐cm and 6‐cm tissue culture plates
  • 4‐ml polystyrene tubes (Falcon)
  • 24‐well and 6‐well tissue culture plates
NOTE: All tissue culture incubations are performed in a humidified 37°C, 5% CO 2 incubator.
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Figures

Videos

Literature Cited

Literature Cited
   Barinaga, M. 1994. Researchers devise a master gene control switch. Science 265:26‐28.
   Blau, H.M. and Rossi, F.M.V. 1999. Tet B or not tet B: Advances in tetracycline‐inducible gene expression. Proc. Natl. Acad. Sci. U.S.A. 96:797-799.
   Chen, J., Kelz, M.B., Zeng, G., Steffen, C., Shockett, P.E., Terwilliger, G., Schatz, D.G., and Nestler, E.J. 2002. Inducible, reversible hair loss in transgenic mice. Transgenic Res. 11:241–247.
   Damke, H., Gossen, M., Freundlieb, S., Bujard, H., and Schmid, S.L. 1995. Tightly regulated and inducible expression of dominant interfering dynamin mutant in stably transformed HeLa cells. Methods Enzymol. 257:209‐220.
   Gossen, M. and Bujard, H. 1992. Tight control of gene expression in mammalian cells by tetracycline‐responsive promoters. Proc. Natl. Acad. Sci. U.S.A. 89:5547‐5551.
   Gossen, M., Bonin, A.L., and Bujard, H. 1993. Control of gene activity in higher eukaryotic cells by prokaryotic regulatory elements. Trends Biochem. Sci. 18:471‐475.
  Sheehy, A.M. and Schlissel, M.S. 1999. Overexpression of RelA causes G1 arrest and apoptosis in a Pro‐B cell line. J. Biol. Chem. 274:8708–8716.
   Shockett, P.E. and Schatz, D.G. 1996. Commentary: Diverse strategies for tetracycline‐regulated inducible gene expression. Proc. Natl. Acad. Sci. U.S.A. 93:5173‐5176.
   Shockett, P., Difilippantonio, M., Hellman, N., and Schatz, D. 1995. A modified tetracycline‐regulated system provides autoregulatory, inducible gene expression in cultured cells and transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 92:6522‐6526.
   Shockett, P.E., Zhou, S., Hong, X., and Schatz, D.G. 2004. Partial reconstitution of V(D)J rearrangement and lymphocyte development in RAG‐deficient mice expressing inducible, tetracycline‐regulated RAG transgenes. Mol. Immunol. 40:813–829.
   Sikes, M.L., Suarez, C.C., and Oltz, E.M. 1999. Regulation of V(D)J recombination by transcriptional promoters. Mol. Cell Biol. 19:2773–2781.
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