DNA Cassette Exchange in ES Cells Mediated by FLP Recombinase: An Efficient Strategy for Repeated Modification of Tagged Loci by Marker-Free Constructs ² Jost Seibler, ‡,⊥ Dirk Schu ¨beler, ‡,⊥ Steven Fiering, § Mark Groudine, | and Ju ¨rgen Bode* ,‡ GBF, National Research Center for Biotechnology, Mascheroder Weg 1, D-38124 Braunschweig, Dartmouth Medical School, Lebanon, New Hampshire 07356, and Fred Hutchinson Cancer Research Center, Seattle, Washington 98104 ReceiVed February 4, 1998; ReVised Manuscript ReceiVed March 13, 1998 ABSTRACT: 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. The availability of techniques for generating transgenic mice from genetically modified embryonic stem cells provides a means to analyze specific gene functions or cis- regulatory DNA elements in normally developing mammals. So far, most studies have created null mutations in order to assay the function of a gene product. During such a “knockout” experiment a coding region is replaced or disrupted by a selectable marker gene using homologous recombination techniques. Current extensions of this ap- proach are aimed at more subtle, multiple and independent changes either in the gene product or the DNA sequences regulating the locus, a concept referred to as a “knock-in”. Efficient repetitive modification of a particular locus is desired not only in association with HR to introduce subtle mutations in a known locus, but also for randomly integrated constructs where the expression characteristics of each integration site can be exploited to express transgenes in a defined and predictable manner. So far, the study of cis- regulatory elements that are randomly integrated into cell lines has been limited by the powerful effects of genomic position on transcription of the integrated construct. Tran- sient transfection, the available alternative, is clearly unable to analyze elements that are functional only after integration into the genome (1). Every advanced strategy for introducing secondary modifications into predefined loci is based on first tagging the locus with a selectable marker. Once the initial tag has been inserted there are systems available that facilitate secondary modifications to the site, but each of these systems has certain limitations. Limitations of Present Systems and How They Are Remedied. Two types of procedures have been described for repetitive modifications of genomic loci; one is based on HR and the other on site-specific recombination (SR). In the first case, the efficiency is limited by the frequency of the HR 1 which differs widely among loci and cell lines. Moreover, common procedures such as the “plug-and-socket” approach (2) leave behind a selectable marker causing a potential problem since an increasing number of examples is reported where the presence of an expressed selectable marker deregulates a locus (3-8 and references therein). Finally, HR cannot be used for modifying sites tagged by random integration unless these have been cloned and characterized. Published methods based on SR circumvent some of these limitations. Up to now, however, all SR-based systems also leave behind a selectable marker, and if a single recombinase site is used as a target, unwanted vector sequences are inserted. The technical goal we have pursued here is the ² This work was supported by grants from Deutsche Forschungsge- meinschaft to J.B. (Bo 419/5-2), from the NIH (CA54337 and DK44746) to M.G., from the American society of hematology and the Burroughs-Wellcome Foundation to S.F. and a career development grant from the Fonds der Chemischen Industrie to D.S. * To whom correspondence should be addressed. ‡ Gesellschaft fu ¨r Biotechnologische Forschung. ⊥ Both authors contributed equally to this work. § Dartmouth Medical School. | Fred Hutchinson Cancer Research Center. 1 Abbreviations: FRT, FLP-recombinase target site; GANC, gan- ciclovir; HR, homologous recombination; HYG, hygromycin; RMCE, recombinase-mediated cassette exchange; SR, site-specific recombina- tion. 6229 Biochemistry 1998, 37, 6229-6234 S0006-2960(98)00288-8 CCC: $15.00 © 1998 American Chemical Society Published on Web 04/16/1998