A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse

Nat Biotechnol. 2003 May;21(5):562-5. doi: 10.1038/nbt811. Epub 2003 Mar 31.

Abstract

Functional redundancies, compensatory mechanisms, and lethal phenotypes often prevent the full analysis of gene functions through generation of germline null mutations in the mouse. The use of site-specific recombinases, such as Cre, which catalyzes recombination between loxP sites, has allowed the engineering of mice harboring targeted somatic mutations, which are both temporally controlled and cell-type restricted. Many Cre-expressing mouse lines exist, but only a few transgenic lines are available that harbor a reporter gene whose expression is dependent on a Cre-mediated event. Moreover, their use to monitor gene ablation at the level of individual cells is often limited, as in some tissues the reporter gene may be silenced, be affected by position-effect variegation, or reside in a chromatin configuration inaccessible for recombination. Thus, one cannot validly extrapolate from the expression of a reporter transgene to an identical ablation pattern for the conditional allele of a given gene. By combining the ability of Cre recombinase to invert or excise a DNA fragment, depending on the orientation of the flanking loxP sites, and the availability of both wild-type (WT) and mutant loxP sites, we designed a Cre-dependent genetic switch (FLEx switch) through which the expression of a given gene is turned off, while the expression of another one is concomitantly turned on. We demonstrate the efficiency and reliability of this switch to readily detect, in the mouse, at the single cell level, Cre-mediated gene ablation. We discuss how this strategy can be used to generate genetic modifications in a conditional manner.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't
  • Technical Report

MeSH terms

  • Animals
  • COS Cells
  • Gene Expression Profiling / methods*
  • Gene Expression Regulation / genetics*
  • Gene Silencing*
  • Integrases / analysis
  • Integrases / genetics*
  • Integrases / metabolism*
  • Mice
  • Mice, Transgenic / genetics
  • Mice, Transgenic / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Viral Proteins / analysis
  • Viral Proteins / genetics*
  • Viral Proteins / metabolism*

Substances

  • Recombinant Proteins
  • Viral Proteins
  • Cre recombinase
  • Integrases