Related Experiment Video
Updated: Jul 7, 2026
08:21
Site-specific Bacterial Chromosome Engineering: ΦC31 Integrase Mediated Cassette Exchange (IMCE)
Published on: March 16, 2012
Bacteriophage P2 integrase: another possible tool for site-specific recombination in eukaryotic cells
C Frumerie1, L Sylwan, T Helleday
1Department of Genetics, Microbiology and Toxicology, Stockholm University, Stockholm, Sweden.
Journal of Applied Microbiology
|February 21, 2008
Summary
The P2 integrase (Int) protein facilitates site-specific gene integration into human DNA, even without bacterial factors. This phage protein shows nuclear localization, suggesting its potential for gene therapy applications.
Area of Science:
- Molecular Biology
- Genetics
- Virology
Background:
- Site-specific recombination is crucial for gene integration.
- Bacterial integrases often require host factors like Integration Host Factor (IHF).
- Phage integrases are potential tools for gene transfer but require evaluation in eukaryotic systems.
Purpose of the Study:
- To assess the potential of phage P2's integrase (Int) for eukaryotic gene transfer.
- To determine if P2 Int can recombine with non-phage sequences and bypass IHF dependency.
- To investigate the nuclear localization of P2 Int in eukaryotic cells.
Main Methods:
- Site-specific recombination assays in E. coli and in vitro using human DNA sequences.
- Modification of phage attachment sites with high-mobility group recognition boxes.
- Fluorescence microscopy to track Int protein localization in HeLa and 293 cells.
Main Results:
- P2 Int catalyzed recombination with a human sequence, albeit less efficiently than with bacterial sequences.
- Modified attachment sites abolished IHF requirement, enabling efficient recombination in eukaryotic cell extracts.
- P2 Int demonstrated functional nuclear localization in human cell lines.
Conclusions:
- P2 Int is a promising candidate for site-specific gene integration into the human genome.
- Prokaryotic integrases offer versatile tools for gene therapy, enabling targeted integration at diverse sites.
Related Concept Videos
02:53Conservative Site-specific Recombination and Phase Variation
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
The recognition sites for Cre recombinase called LoxP...
01:26DNA Bacteriophages
Bacteriophages, or phages, are viruses that specifically infect bacteria, utilizing their genetic material to hijack host cellular machinery for replication. DNA bacteriophages employ single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) genomes. These phages exhibit diverse replication strategies and host interactions, influencing their ecological roles and applications in biotechnology and medicine.ssDNA BacteriophagesssDNA phages, with their small genomes, utilize unique strategies to...
00:57Viral Recombination
Cells are sometimes infected by more than one virus at once. When two viruses disassemble to expose their genomes for replication in the same cell, similar regions of their genomes can pair together and exchange sequences in a process called recombination. Alternatively, viruses with segmented genomes can swap segments in a process called reassortment.
02:53CRISPR and crRNAs
Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
02:31Homologous Recombination
The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
00:43Lysogenic Cycle of Bacteriophages
In contrast to the lytic cycle, phages infecting bacteria via the lysogenic cycle do not immediately kill their host cell. Instead, they combine their genome with the host genome, allowing the bacteria to replicate the phage DNA along with the bacterial genome. The incorporated copy of the phage genome is called the prophage. Some prophages can re-activate and enter the lytic cycle. This often occurs in response to a perturbation, such as DNA damage, but can also transpire in the absence of...