Recombination-mediated Cassette Exchange
Recombination-mediated Cassette Exchange - Related Videos
11:36
Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus
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Cited by 3 •
2016
Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.
08:21
Site-specific Bacterial Chromosome Engineering: ΦC31 Integrase Mediated Cassette Exchange (IMCE)
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Cited by 11 •
2012
A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the ΦC31 integrase.
15:13
Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange
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Cited by 6 •
2017
Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.
09:38
Site-Directed φC31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria
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Cited by 3 •
2021
The protocol describes how to achieve site-directed modifications in the genome of Anopheles malaria mosquitoes using the φC31 system. Modifications described include both the integration and the exchange of transgenic cassettes in the genome of attP-bearing docking lines.
08:56
Recombineering and Gene Targeting
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2023
One of the most widely used tools in modern biology is molecular cloning with restriction enzymes, which create compatible ends between DNA fragments that allow them to be joined together. However, this technique has certain restrictions that limit its applicability for large or complex DNA construct generation. A newer technique that addresses some of these shortcomings is recombineering, which modifies DNA using homologous recombination (HR), the exchange between different DNA molecules based...
FAQs
The resident and donor cassettes are flanked by matching DNA recognition sites that are identified by the recombinase. These sites provide the sequence-specific landmarks for strand cleavage, rearrangement, and re-ligation. Because the exchange is directed by defined boundaries, the incoming cassette is placed at the intended genomic or plasmid location rather than being broadly rearranged throughout surrounding DNA.
The recombinase catalyzes the molecular operations that make the exchange possible. After recognizing the paired DNA sites, it promotes strand cleavage, rearranges the participating DNA segments, and supports their re-ligation. This coordinated activity converts a resident-cassette configuration into one containing the donor cassette, while preserving the site-directed nature of the engineering strategy.
A predetermined site helps separate the effect of the inserted cassette from variability caused by different integration locations. Recombination-mediated Cassette Exchange can therefore support consistent transgene placement and expression across engineered cell lines or organisms. This consistency is especially useful when researchers compare gene variants or regulatory elements and want location to remain a controlled feature.
Its distinguishing feature is the use of defined recognition sites and a recombinase-directed exchange rather than an uncontrolled change across surrounding DNA. The resident and donor sequences are organized around those boundaries, allowing the intended cassette to be replaced at a selected genomic or plasmid location. The approach consequently emphasizes controlled, localized modification.
A basic setup contains a resident DNA cassette, a donor cassette, matching recognition sites flanking the relevant sequences, and the recombinase that catalyzes exchange. The target can be a genomic or plasmid location. Together, these components establish the substrate, the replacement sequence, the recognition boundaries, and the catalytic activity required for site-specific rearrangement.
The method is useful when several transgenes, gene variants, or regulatory elements need to be evaluated at the same engineered location. Replacing one cassette with another reduces variation in placement between constructs, making comparisons more systematic. In this context, the exchange serves as an experimental framework for testing how sequence changes influence transgene expression or other functional outcomes.
In biology, the strategy supports functional genomics, reporter construction, and disease modeling. It also enables systematic comparison of regulatory elements and gene variants by providing a shared genomic or plasmid context for different cassettes. These applications use the method's controlled placement to connect a defined genetic change with an interpretable experimental comparison.
For reporter construction, different reporter or regulatory cassettes can be exchanged at a predetermined location, helping researchers compare their behavior under a consistent placement scheme. In disease modeling, alternative gene variants or related engineered sequences can occupy the same genomic or plasmid site. This design supports controlled functional studies rather than comparisons confounded by different insertion locations.