Transgenic Research

, Volume 20, Issue 3, pp 709–720

Development of a BAC vector for integration-independent and tight regulation of transgenes in rodents via the Tet system

  • Kai Schönig
  • David Kentner
  • Manfred Gossen
  • Tina Baldinger
  • Jun Miao
  • Katrin Welzel
  • Andreas Vente
  • Dusan Bartsch
  • Hermann Bujard
Technical Report

Abstract

The establishment of functional transgenic mouse lines is often limited by problems caused by integration site effects on the expression construct. Similarly, tetracycline (Tet) controlled transcription units most commonly used for conditional transgene expression in mice are strongly influenced by their genomic surrounding. Using bacterial artificial chromosome (BAC) technology in constitutive expression systems, it has been shown that integration site effects resulting in unwanted expression patterns can be largely eliminated. Here we describe a strategy to minimize unfavourable integration effects on conditional expression constructs based on a 75 kb genomic BAC fragment. This fragment was derived from a transgenic mouse line, termed LC-1, which carries the Tet-inducible genes luciferase and cre (Schönig et al. 2002). Animals of this mouse line have previously been shown to exhibit optimal expression properties in terms of tightness in the off state and the absolute level of induction, when mated to appropriate transactivator expressing mice. Here we report the cloning and identification of the transgenic LC-1 integration site which was subsequently inserted into a bacterial artificial chromosome. We demonstrate that this vector facilitates the efficient generation of transgenic mouse and rat lines, where the Tet-controlled expression unit is shielded from perturbations caused by the integration site.

Keywords

Tet system BAC LC-1 Inducible expression Doxycycline 

Supplementary material

11248_2010_9427_MOESM1_ESM.tif (7.5 mb)
Supplementary Fig. 1 Luciferase activity in livers of E11 1-5 animals in the induced and uninduced state (TIFF 7642 kb)
11248_2010_9427_MOESM2_ESM.tif (7.8 mb)
Supplementary Fig. 2 Schematic outline of the genomic region cloned in BAC E11 including S/MARs (TIFF 7973 kb)

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Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Kai Schönig
    • 1
  • David Kentner
    • 2
  • Manfred Gossen
    • 3
  • Tina Baldinger
    • 4
  • Jun Miao
    • 5
  • Katrin Welzel
    • 6
  • Andreas Vente
    • 6
  • Dusan Bartsch
    • 1
  • Hermann Bujard
    • 2
  1. 1.Department of Molecular BiologyCentral Institute of Metal Health and Heidelberg University, Medical Faculty MannheimMannheimGermany
  2. 2.Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH)HeidelbergGermany
  3. 3.Berlin-Brandenburg Center for Regenerative TherapiesBerlinGermany
  4. 4.Klinik und Poliklink für NeurologieCharité -UniversitätsmedizinBerlinGermany
  5. 5.Department of EntomologyThe Pennsylvania State UniversityUniversity ParkUSA
  6. 6.Deutsches Ressourcenzentrum für Genomforschung, RZPDBerlinGermany

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