recombinases. from tag-and-target to tag-and ...

University of Debrecen, Hungary 19th August 2013 – 23rd August 2013 76 F6.

SITE-SPECIFIC RECOMBINASES. FROM TAG-AND-TARGET TO TAG-AND-EXCHANGEBASED GENOMIC MODIFICATIONS Jürgen Bode1, Klar, M.2, Qiao, J.3, Kuehle, J.1 and Turan, S.1 Medical School (MHH) / Institute of Experimental Haematology; D - 30625 Hannover; 2Charité Universitätsmedizin Berlin / Molecular Neonatology; D-13353 Berlin; 3WuXi AppTec Co., Shanghai; 206131 China The advance of Tyr-recombinases Flp and Cre in the early 1990ies opened novel options for the predictable association of replicating nonviral minicircles with open chromatin structures [1] or the predictable insertion of transgenes into compatible target sites of the mammalian host genome [2]. Both efforts were greatly supported by biomathematical (SIDD-) procedures to analyze the DNA strand separation potential of participating sequences localized both at the genomic target site and on the incoming vector [3]. The spectrum of applications could be further expanded by Flp-Recombinase Mediated Cassette Exchange (RMCE) approaches in 1994 [reviewed in 2]. RMCE enables refined tag-and-exchange strategies to replace an existing target structure by a compatible donor cassette devoid of auxiliary vector parts. Such a “gene swap” safely locks the incoming cassette which, however, can be re-mobilized and exchanged at will in case other compatible donors are provided (“serial RMCE”). During the first decade these features considerably enhanced the potential of systematic genome modifications. Based on the reproducible expression capacity of the resulting cell strains and the availability of mutiple heterospecific FRT mutants, a comprehensive toolbox [4] emerged to serve a wide spectrum of purposes [5]. While the concept guaranteed predictable expression characteristics [6], it approached, but did not reach the production levels of industrial multiplecopy cell lines. Its exceptional potential emerged, however, once it entered the stem cell field. Here RMCE has started to contribute to the reliable generation of induced pluripotent stem (iPS-) cells and their subsequent differentiation to a variety of cell types for diagnostic and therapeutic purposes. An unanticipated property concerns the capacity to enable serial RMCE reactions [7] even after the reprogramming cassette underwent the obligatory silencing process. During this phase of Flp-induced recombination steps chromatin re-opening is associated with the loss of heterochromatic CpG methylationand H3K9me3-marks. With these properties in mind we have added composite Flp-recognition target sites (“heterospecific FRT-doublets”), to the LTRs of lentiviral vectors. These “twin sites” enhance the safety of iPS re-programming and –differentiation as they enable the subsequent quantitative excision of transgenes, leaving behind a highly accessible,single “FRT-twin” [7]. This composite element represents a unique genomic tag, serving to characterize the host locus in order to identify genomic targets meeting the recent stringent Genomic Safe Harbor criteria. These tagged „GSHs” lend themselves to multiple dedicated (re)uses, solely based on the potential of the actual Flp-RMCE repository. [1.] Nehlsen K et al. (2013) In "Minicircle and Plasmid DNA Vectors - The Future of non-viral and viral GeneTransfer" pp115-162, ISBN 978-3-527-32456-9 - Wiley-VCH, Weinheim . [2.] Turan S & Bode J (2011) FASEB J. 25, 4088-4107 http://dx.doi.org/10.1096/fj.11-186940 [3.] Klar M & Bode J (2005) Mol. Cell. Biol. 25, 10159-10170. http://dx.doi.org/10.1128/MCB.25.22.1015910170.2005 [4.] Bode J et al. Biol. Chem. 381:801-813. http://dx.doi.org/10.1515/BC.2000.103 [5.] Turan S et al. (2010) J. Mol. Biol. 402 , 52-69. http://dx.doi.org/10.1016/j.jmb.2010.07.015 [6.] Qiao J ert al. (2009) J. Mol. Biol. 390, 579–594. http://dx.doi.org/10.1016/j.jmb.2009.05.012 . [7.] Turan S et al. (2013) Gene 515, 1–27. http://dx.doi.org/10.1016/j.gene.2012.11.016 1Hannover

This work was supported by the Excellence Initiative REBIRTH (Regenerative Biology to Reconstructive Therapy), the SFB 738 (Optimization of conventional and innovative transplants