The transgeneticist's toolbox: Novel methods for the targeted modificationof eukaryotic genomes

Classical techniques for gene transfer into mammalian cells involve tedious screening procedures to identify transgenic clones or animals with the app ropriate level and stability of expression or with the correct developmenta l patterns. These first generation technologies are clearly inadequate for complex genetic strategies by which gene regulation can be studied in its e ntire complexity While site-specific insertions can principally be achieved by homologous recombination or by adapting the recombination apparatus fro m phages or yeast, these methods usually lack the required efficiency or th ey perturb expression patterns by the co-insertion of prokaryotic vector pa rts. Virtually all of these problems can be overcome by recombinase-mediate d cassette exchange (RMCE) techniques which cleanly replace a resident cass ette that is flanked by two hetero-specific recombination target sites for a second cassette with the analogous design, presented on a targeting vecto r. After illustrating the fundamentals of site-specific recombination by se lected experiments, the authors (arranged in the chronological order of the ir contribution) will describe their efforts to develop RMCE into a method of wide applicability. Further developments that have been initiated utiliz ing the particular potential of the RMCE principle will be outlined.