Use of Mutated FLP Recognition Target (FRT) Sites for the Exchange of Expression Cassettes at Defined Chromosomal Loci

Schlake, Thomas; Bode, Jürgen

doi:10.1021/bi00209a003

Cited by 275 publications

(296 citation statements)

References 15 publications

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“…This process leads to the exchange of the positive-negative selection marker (+/À) for a gene variant (Gene*) to yield a modified locus (T¢¢). These processes have been described in detail before (Schlake and Bode 1994;Seibler et al 1998;reviews: Bode et al 2000reviews: Bode et al , 2003Baer and Bode 2001).…”

Section: Introductionmentioning

confidence: 99%

“…While these steps are usually separated in time for HR, for SR there is little if any evidence for longlived intermediates (crossover at one or the other target site in SR, see Schlake and Bode 1994). This is the rationale for our simplified representation of RMCE (immediate replacement of one cassette for another by double-crossover) in all figures.…”

Section: Introductionmentioning

confidence: 99%

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Recommended Method for Chromosome Exploitation: RMCE-based Cassette-exchange Systems in Animal Cell Biotechnology

et al. 2006

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The availability of site-specific recombinases has revolutionized the rational construction of cell lines with predictable properties. Early efforts were directed to providing pre-characterized genomic loci with a single recombinase target site that served as an address for the integration of vectors carrying a compatible tag. Efficient procedures of this type had to await recombinases like UC31, which recombine attP and attB target sites in a one-way reaction -at least in the cellular environment of the higher eukaryotic cell. Still these procedures lead to the co-introduction of prokaryotic vector sequences that are known to cause epigenetic silencing. This review illuminates the actual status of the more advanced recombinase-mediated cassette exchange (RMCE) techniques that have been developed for the major members of site-specific recombinases (SR), Flp, Cre and UC31. In RMCE the genomic address consists of a set of heterospecific recombinase target (RT-) sites permitting the exchange of the intervening sequence for the gene of interest (GOI), as part of a similar cassette. This process locks the GOI in place and it is 'clean' in the sense that it does not co-introduce prokaryotic vector parts nor does it leave behind a selection marker.Abbreviations: FRT -Flp-recombinase target site; HR -homologous recombination; RMCE -recombinase-mediated cassette exchange; RT -recombinase target; SR -site-specific recombination

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Recommended Method for Chromosome Exploitation: RMCE-based Cassette-exchange Systems in Animal Cell Biotechnology

et al. 2006

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“…These four PG368 clones were subsequently targeted with pEMTAR.MFGeGFP, 19 a plasmid containing the Fn and F5 recombination sites (FTR sites 24 ) and an LTRdriven MFG-based retroviral vector 25 with enhanced green fluorescent protein (eGFP) as a marker gene and an IRES-coupled 'atg' start codon downstream of the 3 0 LTR (Figure 1a). Cells were cotransfected with a plasmid encoding for the Flp-recombinase to allow site-specific recombination at the FRT sites.…”

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confidence: 99%

A new PG13-based packaging cell line for stable production of clinical-grade self-inactivating γ-retroviral vectors using targeted integration

et al. 2009

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The clinical application of self-inactivating (SIN) retroviral vectors has been hampered by the lack of reliable and efficient vector production technologies. To enable production of SIN g-retroviral vectors from stable producer clones, a new PG13-based packaging cell, known as PG368, was developed. Viral vector expression constructs can be reliably inserted at a predefined genomic locus of PG368 packaging cells by an Flp-recombinasemediated targeted cassette exchange (RMCE) reaction. A new, carefully designed vector-targeting construct, pEMTAR-1, eliminated the co-packaging of the selectable marker gene used for the identification of successful recombination at the predefined genomic locus and thus, improved the safety of the production system. Selected clones produced vector supernatants at consistent titers. The targeted insertion of therapeutically relevant SIN vectors for chronic granulomatous disease and X-linked severe combined immunodeficiency into PG368 cells results in stable titers within the range necessary for clinical application. The production of retroviral SIN vectors from stable clinical-grade producer cells is feasible and will contribute to the safe production and application of SIN g-retroviral vectors for clinical trials.

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“…This clearly emphasizes the importance of the final chromatin structure for the action of these elements. (iii) SAR elements forming an efficient mini domain by flanking a gene at either end cooperate, frequently in a synergistic manner (42,61).…”

Section: Discussionmentioning

confidence: 99%

A plant scaffold attached region detected close to a T-DNA integration site is active in mammalian cells

et al. 1994

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Integration of foreign genes into plant genomes by the Agrobacterlum T-DNA transfer system has been considered to occur at random. It has been speculated that the chromosomal structure of the integration site might affect the expression pattern of the introduced genes. To gain insight into the molecular structure of T-DNA integration sites and its possible impact on gene expression, we have examined plant DNA sequences In the vicinity of T-DNA borders. Analysis of a transgenic petunia plant containing a chloramphenicol acetyltransferase (CAT) gene regulated by the hemoglobin promoter (PAR) from Parasponia andersonii revealed a scaffold attachment region (SAR) close to one T-DNA end. In addition to having strong binding affinities for both animal and plant nuclear scaffolds this petunia SAR element is as active in mammalian cells as the authentic elements from mammalian sources.

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Use of Mutated FLP Recognition Target (FRT) Sites for the Exchange of Expression Cassettes at Defined Chromosomal Loci

Cited by 275 publications

References 15 publications

Recommended Method for Chromosome Exploitation: RMCE-based Cassette-exchange Systems in Animal Cell Biotechnology

Recommended Method for Chromosome Exploitation: RMCE-based Cassette-exchange Systems in Animal Cell Biotechnology

A new PG13-based packaging cell line for stable production of clinical-grade self-inactivating γ-retroviral vectors using targeted integration

A plant scaffold attached region detected close to a T-DNA integration site is active in mammalian cells

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