Phic31 Integrase
Phic31 Integrase - Related Videos
04:21
phiC31-Integrase-Mediated Site-Directed Transgene Integration: A Microinjection Technique for Site-Specific Transgene Integration into an Anopheles Vector
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2025
In this video, we demonstrate phiC31-mediated site-specific transgene integration in the Anopheles embryo via microinjection. This method helps introduce desired genes into a host system for research and industrial applications.
03:45
Purification of Viral Integrase Using Affinity Chromatography
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2026
Source: Lopez Jr., M. A., et al. Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase. J. Vis. Exp. (2017)This video demonstrates the purification of polyhistidine-tagged viral integrase using nickel affinity chromatography. The protocol uses imidazole gradient elution to selectively isolate the integrase based on its affinity for nickel-charged resin.
02:21
Purification of Viral Integrase Using Heparin Affinity Chromatography
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2026
Source: Lopez Jr., M. et al. Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase. J. Vis. Exp. (2017)This video demonstrates the procedure for purifying viral integrase from bacterial nuclease contamination using heparin Sepharose column chromatography followed by SDS-PAGE analysis to confirm protein purity.
07:50
Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase
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Cited by 10 •
2017
Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.
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.
FAQs
PhiC31 integrase brings the attB and attP DNA sites together and catalyzes their exchange. The reaction produces two new junctions, attL and attR, rather than preserving the original site pair. This change in site identity helps make the integration stable and generally unidirectional, supporting long-term maintenance of an inserted genetic sequence.
A generally unidirectional reaction favors movement from the attB and attP arrangement toward the attL and attR products. In bioengineering, that behavior helps an integrated transgene remain in place instead of readily returning to its starting configuration. The resulting stability is particularly useful for gene-expression studies and for engineering mammalian cell lines.
PhiC31 integrase does not require extensive stretches of matching DNA between an inserted construct and its chromosomal target. Instead, it relies on the defined attB and attP recognition sequences. This distinction can simplify the sequence requirements for stable insertion and provides an alternative to approaches that depend on long regions of homology.
The attB and attP sequences serve as the recognition points that specify where the recombination reaction can occur. Their presence gives the integrase a relatively simple molecular address for joining an engineered construct with a DNA target. After recombination, their conversion into attL and attR records that the integration event has taken place.
A construct is designed with an attB sequence, while the intended DNA target contains an attP sequence or is engineered to provide one. PhiC31 integrase is then supplied in the biological system so it can catalyze recombination between the sites. The expected outcome is formation of attL and attR together with stable insertion of the transgene.
PhiC31 integrase supports several applications that require genetic material to persist in cells. In mammalian systems, these include stable transgene integration, controlled gene-expression studies, and cell-line engineering. It is also relevant to experimental gene-therapy development, where a site-specific integration mechanism and limited homology requirements can be advantageous.