Site-specific Recombination in Eukaryotic Cells Mediated by Mutant λ Integrases: Implications for Synaptic Complex Formation and the Reactivity of Episomal DNA Segments

Christ, Nicole; Corona, Teresa; Dröge, Peter

doi:10.1016/s0022-2836(02)00327-3

Cited by 29 publications

(40 citation statements)

References 22 publications

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“…As shown before, wild-type Int is only marginally active in integrative and excisive recombination on episomal substrates in HeLa cells (13). By applying the same test system, i.e.…”

Section: Scihf2 Triggers L Int-mediated Recombination In Human Cellsmentioning

confidence: 56%

“…8, lower right panel). Further, the activities of mutant integrases, Int-h and Int-h/218, which already catalyze both reactions in the absence of IHF with high ef®ciency (13), are not signi®cantly affected by the presence of scIHF2 (Fig. 8, middle panels).…”

Section: Scihf2 Triggers L Int-mediated Recombination In Human Cellsmentioning

confidence: 96%

“…A PGK-puromycin R -PA fragment was subsequently inserted into the blunted SapI site of the resulting vector. Substrate vectors for integrative and excisive recombination containing attB/attP (plIR) or attL/attR (plER) as direct repeats have been described (13). pNC9WT is a derivate of pNC9 (17) and was generated via PCR.…”

Section: Dna Constructsmentioning

confidence: 99%

“…However, we also demonstrated that puri®ed IHF pre-bound to a DNA substrate for integrative recombination and transfected into mammalian cells stimulates wildtype Int-mediated recombination. This result revealed an interesting possibility of regaining control over the directionality of recombination in mammalian cells through the employment of protein cofactors and wild-type Int (13). However, it is known that separate over-expression of IHF a-and b-subunits in E.coli results in unstable polypeptides and insoluble aggregates (14,15).…”

Section: Introductionmentioning

confidence: 99%

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Activation of site-specific DNA integration in human cells by a single chain integration host factor

Corona

Bao²,

Christ³

et al. 2003

Nucleic Acids Research

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The heterodimeric integration host factor (IHF) is a site-speci®c DNA-binding and DNA-bending protein from Escherichia coli. It plays essential roles in a variety of DNA transactions including recombination, transcription and DNA replication. IHF's ability for concerted binding and bending of DNA is key to its biological function. Here we report the design, characterization and application of a single polypeptide chain IHF, termed scIHF2. In a novel approach for protein engineering, we inserted almost the entire a-subunit of IHF into the b-subunit. DNA binding and DNA bending assays revealed that puri®ed wild-type IHF and scIHF2 behave very similarly. Further, scIHF2 is required for site-speci®c integrative recombination by phage l integrase and for pSC101 replication in a DIHF E.coli host. It also triggers site-speci®c integrative and excisive recombination in vitro to the same extent as the wild-type protein. We also demonstrate that scIHF2 is stably expressed in HeLa cells, that it is localized primarily in the cell nucleus and that it triggers integrative recombination in mammalian cells by wild-type integrase. Hence, scIHF2 may be used as a novel regulatory cofactor for recombination or other DNA transactions in mammalian cells that require or bene®t from sequence-speci®c high precision DNA bending.

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“…As shown before, wild-type Int is only marginally active in integrative and excisive recombination on episomal substrates in HeLa cells (13). By applying the same test system, i.e.…”

Section: Scihf2 Triggers L Int-mediated Recombination In Human Cellsmentioning

confidence: 56%

Section: Scihf2 Triggers L Int-mediated Recombination In Human Cellsmentioning

confidence: 96%

Section: Dna Constructsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Activation of site-specific DNA integration in human cells by a single chain integration host factor

Corona

Bao²,

Christ³

et al. 2003

Nucleic Acids Research

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“…The weaker properties of FLP, as well as the merits in finding a third efficient recombinase for genome engineering, prompted searches for new tyrosine SSR tools (Araki et al, 1992;Ringrose et al, 1997;Christ et al, 2002 …”

Section: Introductionmentioning

confidence: 99%

Dre recombinase, like Cre, is a highly efficient site-specific recombinase in E. coli, mammalian cells and mice

Anastassiadis

Patsch

et al. 2009

Disease Models &Amp; Mechanisms

256

203

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SUMMARY Tyrosine site-specific recombinases (SSRs) including Cre and FLP are essential tools for DNA and genome engineering. Cre has long been recognized as the best SSR for genome engineering, particularly in mice. Obtaining another SSR that is as good as Cre will be a valuable addition to the genomic toolbox. To this end, we have developed and validated reagents for the Dre-rox system. These include an Escherichia coli-inducible expression vector based on the temperature-sensitive pSC101 plasmid, a mammalian expression vector based on the CAGGs promoter, a rox-lacZ reporter embryonic stem (ES) cell line based on targeting at the Rosa26 locus, the accompanying Rosa26-rox reporter mouse line, and a CAGGs-Dre deleter mouse line. We also show that a Dre-progesterone receptor shows good ligand-responsive induction properties. Furthermore, we show that there is no crossover recombination between Cre-rox or Dre-loxP. Hence, we add another set of efficient tools to the genomic toolbox, which will enable the development of more sophisticated mouse models for the analysis of gene function and disease.

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