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

Expression of experimental constructs in mammalian cells or transgenic anim als is difficult to control because it is markedly influenced by position e ffects. This has limited both the analysis of cis-DNA regulatory elements f or transcription and replication, and the physiological analysis of protein s expressed from transgenes. We report here two new methods based on the co ncept of recombinase-mediated cassette exchange (RMCE) to perform site-spec ific chromosomal integration. The first method permits the exchange of a ne gative selectable marker pre-localized on the chromosome with a transgene v ia a CRE-mediated double recombination between inverted Lox sites. Integrat ion efficiency is close to 100 % of negatively selected mouse erythroleukem ia cells and ranges from 10 to 50 % in embryonic stem cells. The second met hod allows RMCE with no selection at all except for cells that have taken u p 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 re producible expression. RMCE should be useful to develop artificial genetic loci that impart specific and reproducible regulation of transgenes in high er eukaryotes. This should facilitate the analysis of cis-regulatory DNA el ements 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. (C) 1999 Academic Press.