title: Expanding Flp-RMCE options: the potential of Recombinase Mediated Twin-Site Targeting (RMTT).
abstract: Where possible, developments enabling the establishment of cell lines with predictable, long-term stable expression capacity are based on single-copy integrations at safe genomic loci with predictable properties. Robust performance could be assigned to lentiviral transduction systems anchoring single LV-units at sites with adequate transcription potential. In the case of gene therapeutic vectors it is essential that the expression interval can be safely terminated following individual requirements, which has mostly been achieved by lox-mediated excision ("floxing"). To extend the spectrum of possible applications we replaced the common, phage-derived Cre/loxP-setup by modules derived from the yeast "Flp/FRT" site-specific recombination system. This change enables a variety of additional options, for instance by "multiplexing" strategies, which rely on a variety of heterospecific FRT-site variants (F'). If we provide lentiviral LTRs with a "twin-site", here an FF3 fusion, the presence of Flp-recombinase will effectively excise the expression cassette, leaving behind a single neutral, genomically anchored FF3 unit. This tag serves to identify the integration locus and to apply sequence- and structural (SIDD-) analyses to predict its functions. Candidate loci are then used to accommodate, at the given site, other genes of interest by "Recombinase-Mediated Twin Site Targeting" (RMTT), a contemporary extension of existing cassette exchange (RMCE-) routines. Supported by the fact that FF3 twins remain accessible within the host genome, RMTT provides access to certified cell lines as it complies with recently defined stringent genomic safe harbor criteria. Our discussion- and outlook-sections will cover lentiviral targeting strategies and current possibilities to enable their fine-tuning.
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, D-30625 Hannover, Germany; Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.
authors.forename: Soeren
authors.initials: S
authors.lastname: Turan
authors.affiliation: WuXi AppTec Co. Ltd., 288 Fute Zhong Road, Shanghai 200131, China.
authors.forename: Junhua
authors.initials: J
authors.lastname: Qiao
authors.affiliation: Genome and Biomedical Sciences Facility, 451 Health Sciences Drive, Davis, CA 95616, USA.
authors.forename: Sally
authors.initials: S
authors.lastname: Madden
authors.affiliation: UC Davis Genome Center, University of California, One Shields Avenue, Davis, CA 95616, USA.
authors.forename: Craig
authors.initials: C
authors.lastname: Benham
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, D-30625 Hannover, Germany.
authors.forename: Marina
authors.initials: M
authors.lastname: Kotz
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, D-30625 Hannover, Germany; Division of Hematology/Oncology, Children's Hospital Boston, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Axel
authors.initials: A
authors.lastname: Schambach
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, D-30625 Hannover, Germany. Electronic address: bode.juergen@mh-hannover.de.
authors.forename: Juergen
authors.initials: J
authors.lastname: Bode
mesh_terms: D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D012441:Saccharomyces cerevisiae / Q000235:genetics; D020079:Terminal Repeat Sequences; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase
keywords: FRT twin-sites; Genomic safe harbors (GSH); Genomic scout functions; Recombinase Mediated Cassette Exchange (RMCE); Stress-Induced Duplex Destabilization (SIDD)