The transgeneticist's toolbox: novel methods for the targeted modification of eukaryotic genomes.

Classical techniques for gene transfer into mammalian cells involve tedious screening procedures to identify transgenic clones or animals with the appropriate level and stability of expression or with the correct developmental patterns. These first generation technologies are clearly inadequate for complex genetic strategies by which gene regulation can be studied in its entire complexity. While site-specific insertions can principally be achieved by homologous recombination or by adapting the recombination apparatus from phages or yeast, these methods usually lack the required efficiency or they perturb expression patterns by the co-insertion of prokaryotic vector parts. Virtually all of these problems can be overcome by recombinase-mediated cassette exchange (RMCE) techniques which cleanly replace a resident cassette that is flanked by two hetero-specific recombination target sites for a second cassette with the analogous design, presented on a targeting vector. After illustrating the fundamentals of site-specific recombination by selected experiments, the authors (arranged in the chronological order of their contribution) will describe their efforts to develop RMCE into a method of wide applicability. Further developments that have been initiated utilizing the particular potential of the RMCE principle will be outlined.

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

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.

DNA cassette exchange in ES cells mediated by Flp recombinase: an efficient strategy for repeated modification of tagged loci by marker-free constructs.

The repeated modification of a genomic locus is a technically demanding but powerful strategy to analyze the function of a particular gene product or the role of cis-regulatory DNA elements in mammalian cells. The initial step is "tagging" a site with a selectable marker which is done by homologous recombination (HR) to modify a known locus or by random integration to study cis-regulatory elements at a reproducibly accessible genomic location. The tag is then used to target the construct of choice during an exchange step. Presented here is a novel technique in which the exchange is independent of HR and does not introduce vector sequences. It relies on our previous studies on the replacement of DNA cassettes by FLP-recombinase, whereby some common limitations can be overcome. To this end, the tag, a hygtk positive/negative selection marker, is integrated into the genome of embryonic stem (ES) cells. This marker is flanked by a wild-type Flp-recognition target (FRT) site on one end and by a modified heterospecific FRT site on the other. Successful Flp-mediated replacement of the hygtk cassette is enriched by ganciclovir (GANC) selection for cells that lack the encoded fusion protein. Thereby, the hygtk gene can be exchanged for virtually any sequence in a single efficient step without the need of introducing a positive selectable marker. The system can hence be used to analyze the function of either a gene product or regulatory sequences in ES cells or the transgenic mice derived thereof.

Double-reciprocal crossover mediated by FLP-recombinase: a concept and an assay.

FLP recombinase induces a double-reciprocal crossover event between sets of different FLP recognition target (FRT) sites. Therefore, if these sites flank an expression cassette at a given genomic locus, it can be exchanged for another cassette that has been constructed in the analogous was [Schlake & Bode (1994) Biochemistry 33, 12746-12751]. Here we demonstrate that an integrated expression cassette, flanked by a wild type and a mutated site, remains completely stable in the presence of constitutive FLP activity, obviating the need for a timing of this parameter. Therefore the only variable left for optimization is the initial concentration of the exchange plasmid. Since the exchange plasmid lacks a promoter, random integration is not expected to confer resistance to the selection marker, the expression of which requires the acquisition of the SV40 promoter provided at the predetermined integration site. Due to the presence of a luciferase reporter in a specific bicistronic expression cassette, recombination generates bioluminescence upon recombination, indicating the extent of the exchange reaction. This principle is utilized to compare the potential of various cell lines to support the exchange reaction and to adjust the optimum parameters.