Recombinase-Mediated Cassette Exchange (RMCE): Traditional Concepts and Current Challenges

Turan, Soeren; Galla, Melanie; Ernst, Ellen; Jian, Qiao; Voelkel, Christine; Schiedlmeier, Bernhard; Zehe, Christoph; Bode, Jürgen

doi:10.1016/j.jmb.2011.01.004

Cited by 148 publications

(131 citation statements)

References 91 publications

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“…Nevertheless, differences between individual cells or cell clones with regard to the UCOE effect on transgene expression and silencing were observed in our studies. Thus, site-specific integration systems like TALE or zinc finger nucleases and cre-or flip-recombinase-mediated cassette exchange technology [46,47] might be exploited to investigate UCOE-mediated effects on epigenetic modifications at a given locus.…”

Section: Discussionmentioning

confidence: 99%

A ubiquitous chromatin opening element prevents transgene silencing in pluripotent stem cells and their differentiated progeny

et al. 2013

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Methylation‐induced gene silencing represents a major obstacle to efficient transgene expression in pluripotent cells and thereof derived tissues. As ubiquitous chromatin opening elements (UCOE) have been shown to prevent transgene silencing in cell lines and primary hematopoietic cells, we hypothesized a similar activity in pluripotent cells. This concept was investigated in the context of cytidine deaminase (CDD) gene transfer, an approach to render hematopoietic cells resistant to the chemotherapeutic agent Ara‐C. When murine induced pluripotent stem cells (iPSC)/embryonic stem cells (ESCs) were transduced with self‐inactivating lentiviral vectors using housekeeping (truncated elongation factor 1α; EFS) or viral (spleen focus‐forming virus; SFFV) promoters, incorporation of an heterogeneous nuclear ribonucleoproteins A2 B1/chromobox protein homolog 3 locus‐derived UCOE (A2UCOE) significantly increased transgene expression and Ara‐C resistance and effectively prevented silencing of the SFFV‐promoter. The EFS promoter showed relatively stable transgene expression in naïve iPSCs, but rapid transgene silencing was observed upon hematopoietic differentiation. When combined with the A2UCOE, however, the EFS promoter yielded stable transgene expression in 73% ± 6% of CD41+ hematopoietic progeny, markedly increased CDD expression levels, and significantly enhanced Ara‐C resistance in clonogenic cells. Bisulfite sequencing revealed protection from differentiation‐induced promoter CpG methylation to be associated with these effects. Similar transgene promoting activities of the A2UCOE were observed during murine neurogenic differentiation, in naïve human pluripotent cells, and during nondirected multilineage differentiation of these cells. Thus, our data provide strong evidence that UCOEs can efficiently prevent transgene silencing in iPS/ESCs and their differentiated progeny and thereby introduce a generalized concept to circumvent differentiation‐induced transgene silencing during the generation of advanced iPSC/ESC‐based gene and cell therapy products. STEM CELLS2013;31:488–499

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Section: Discussionmentioning

confidence: 99%

A ubiquitous chromatin opening element prevents transgene silencing in pluripotent stem cells and their differentiated progeny

et al. 2013

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“…6B). A recent review of the RMCE reaction discusses the issues involved in optimizing the process, including a comparison of Cre versus Flp recombinases as RMCE enzymes (97).…”

Section: Turning Cre Into An Integrasementioning

confidence: 99%

Cre Recombinase

Duyne

2015

Microbiol Spectr

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The use of Cre recombinase to carry out conditional mutagenesis of transgenes and insert DNA cassettes into eukaryotic chromosomes is widespread. In addition to the numerous in vivo and in vitro applications that have been reported since Cre was first shown to function in yeast and mammalian cells nearly 30 years ago, the Cre-loxP system has also played an important role in understanding the mechanism of recombination by the tyrosine recombinase family of site-specific recombinases. The simplicity of this system, requiring only a single recombinase enzyme and short recombination sequences for robust activity in a variety of contexts, has been an important factor in both cases. This review discusses advances in the Cre recombinase field that have occurred over the past 12 years since the publication of Mobile DNA II. The focus is on those recent contributions that have provided new mechanistic insights into the reaction. Also discussed are modifications of Cre and/or the loxP sequence that have led to improvements in genome engineering applications.

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“…Dre recombinase, a close relative of Cre, has been demonstrated to work in mammalian cells (25, 26). PhiC31 integrase, which catalyzes unidirectional recombination between attP and attB sequences, has been used extensively for integration (27, 28) and cassette exchange (29,30), applications that take advantage of its nonreversibility. The R recombinase, a relative of FLP from the yeast Zygosaccharomyces rouxii (31, 32), has been shown to function in plants (33).…”

Section: And Plants (24)mentioning

confidence: 99%

Multiple new site-specific recombinases for use in manipulating animal genomes

Nern

Pfeiffer

Svoboda

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

154

171

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Site-specific recombinases have been used for two decades to manipulate the structure of animal genomes in highly predictable ways and have become major research tools. However, the small number of recombinases demonstrated to have distinct specificities, low toxicity, and sufficient activity to drive reactions to completion in animals has been a limitation. In this report we show that four recombinases derived from yeast-KD, B2, B3, and R-are highly active and nontoxic in Drosophila and that KD, B2, B3, and the widely used FLP recombinase have distinct target specificities. We also show that the KD and B3 recombinases are active in mice.gene expression | genetic engineering S ite-specific DNA recombinases are widely used in multicellular organisms to manipulate the structure of genomes and, in turn, to control gene expression (for reviews see refs. 1-4). These enzymes, derived from bacteria and fungi, catalyze directionally sensitive DNA exchange reactions between short (30-40 nucleotides) target site sequences that are specific to each recombinase (5). These reactions enable four basic functional modules-excision/insertion, inversion, translocation and cassette exchange-that have been used individually or combined in a wide range of configurations to control gene expression (Fig. 1A).The use of site-specific recombination to manipulate genomes has been limited by the availability of recombinases with high activity, distinct site specificity, and low toxicity. In Drosophila, the most widely used recombinase is FLP, encoded by the Saccharomyces cerevisiae 2-μm plasmid (6). FLP was first shown to work in a heterologous, multicellular organism by Golic and Lindquist in 1989 (7) who demonstrated the excision reaction on chromosomally inserted target sites (FRTs). Since that time FLP/FRT recombination has been widely used in Drosophila in applications based on excision (8) and translocation (9-11).Complex manipulations of genome structure can require the use of more than one of the modules diagrammed in Fig. 1A, or parallel independent implementations of the same module, in a single individual. To accomplish such manipulations, the modules must be implemented with different recombinases that do not recognize each other's target sites. Similarly, a number of powerful methods have been developed for using the excision and inversion reactions to control expression of a transgene specifically in cells where two independent gene expression patterns overlap (2,3,12,13). Such intersectional methods rely on pairs of orthogonal recombinases; see, for example, Fig. 1B. For these reasons, we sought to discover additional recombinases with distinct site-specificity.FLP recombinase has been mutated to recognize altered FRT sites, but some cross-reaction still remains (14, 15). Cre, encoded by the bacteriophage P1, is the most widely used recombinase in mammalian cells (16)(17)(18). Cre functions in Drosophila (19), but exhibits obvious toxicity (20), a problem also observed in mammalian cells (21, 22; reviewed in ref. 23) an...

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Recombinase-Mediated Cassette Exchange (RMCE): Traditional Concepts and Current Challenges

Cited by 148 publications

References 91 publications

A ubiquitous chromatin opening element prevents transgene silencing in pluripotent stem cells and their differentiated progeny

A ubiquitous chromatin opening element prevents transgene silencing in pluripotent stem cells and their differentiated progeny

Cre Recombinase

Multiple new site-specific recombinases for use in manipulating animal genomes

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