Site-specific chromosomal integration in mammalian cells: Highly efficient CRE recombinase-mediated cassette exchange

Yong Qing Feng, Jost Seibler, Raouf Alami, Andrew Eisen, Karen A. Westerman, Philippe Leboulch, Steven Fiering, Eric E. Bouhassira

Research output: Contribution to journalArticle

161 Scopus citations

Abstract

Expression of experimental constructs in mammalian cells or transgenic animals is difficult to control because it is markedly influenced by position effects. This has limited both the analysis of cis-DNA regulatory elements for transcription and replication, and the physiological analysis of proteins expressed from transgenes. We report here two new methods based on the concept of recombinase-mediated cassette exchange (RMCE) to perform site-specific chromosomal integration. The first method permits the exchange of a negative selectable marker pre-localized on the chromosome with a transgene via a CRE-mediated double recombination between inverted Lox sites. Integration efficiency is close to 100% of negatively selected mouse erythroleukemia cells and ranges from 10 to 50% in embryonic stem cells. The second method allows RMCE with no selection at all except for cells that have taken up plasmid transiently. While less efficient, this technique permits novel experimental approaches. We find that integration of a transgene at a given genomic site leads to reproducible expression. RMCE should be useful to develop artificial genetic loci that impart specific and reproducible regulation of transgenes in higher eukaryotes. This should facilitate the analysis of cis-regulatory DNA elements governing expression and position effects, improve our control over the physiological effects of transgenes, and accelerate the development of animal models for complex human diseases.

Original languageEnglish (US)
Pages (from-to)779-785
Number of pages7
JournalJournal of Molecular Biology
Volume292
Issue number4
DOIs
Publication statusPublished - Oct 1 1999

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Keywords

  • CRE/Lox
  • Chromosomal integration
  • Gene transfer
  • RMCE
  • Recombinase

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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