phiC31 integrase, a site-specific recombinase, can effectively mediate foreign genes bearing an attB sequence integrated into pseudo attP sites. We have previously identified two pseudo attP sites, BpsF1 and BpsM1 from the bovine genome. In this study, two new pseudo attP sites, BF4 and BF10, were d

phiC31 integrase, a site-specific recombinase, can effectively mediate foreign genes bearing an attB sequence integrated into pseudo attP sites. We have previously identified two pseudo attP sites, BpsF1 and BpsM1 from the bovine genome. In this study, two new pseudo attP sites, BF4 and BF10, were discovered using half-nested inverse PCR from cow fibroblasts. The genomic locations of these two pseudo attP sites were identified by direct sequencing and a BLAST search, and it was confirmed that they reside at positions 4q31 and 10q35 by fluorescence in situ hybridization analysis. Subsequently, the distinct integration frequencies of the four pseudo attP sites were examined. The BF4 site was identified as a hotspot where site-specific integration occurred in most of the cell clones examined, accounting for 74% (42/57) of the integration; much more than the integration frequency for BF10 (7%; 4/57), BpsF1 (7%; 4/57) and BpsM1 (0/57). Interestingly, similar to other hotspots identified in the human and mouse genomes, in which transgenes integrated at hotspots result in high expression, the GFP gene integrated at hotspot BF4 was expressed at high levels in cow fibroblasts, as confirmed by fluorescence microscopy and FACS analysis. Furthermore, ELISA showed that the expression level of the GFP gene integrated at the BF4 site averaged approximately 328 microg x mg(-1), which is more than twofold higher than that integrated at the BF10 site. This study suggests that somatic cells carrying a desired gene integrated at the BF4 site can be used as nuclear donors to generate valuable transgenic animals by nuclear transfer.

To identify the parts of retroviral integrase that interact with its DNA substrates, we compared the patterns of target site usage by chimeric enzymes and protein fragments in assays that reveal integrase's non-specific nuclease activities. The central region of 12 chimeric proteins between the huma

To identify the parts of retroviral integrase that interact with its DNA substrates, we compared the patterns of target site usage by chimeric enzymes and protein fragments in assays that reveal integrase's non-specific nuclease activities. The central region of 12 chimeric proteins between the human immunodeficiency virus type 1 and visna virus integrases was found to be responsible for selecting non-viral target DNA sites when small alcohols provide the attacking nucleophilic OH group during non-specific alcoholysis assays. Testing deletion derivatives of the integrase protein in this assay, which has similarities to the DNA joining reaction that occurs during retroviral integration, defined a smaller central domain that is sufficient for activity. Thus, this core domain likely contains both the host DNA site and the nucleophile site. Surprisingly, the region of integrase responsible for selecting non-viral target DNA sites when the viral DNA end is the attacking nucleophile could not similarly be mapped with the standard oligonucleotide joining assay. We therefore tested the proteins in a more sensitive assay that displays preferred sites of viral DNA insertion in a plasmid DNA target. All 12 chimeras yielded novel patterns compared with the wild-type enzymes in this assay, although local insertion patterns indicated that the central domain plays an important role in target site selection. Together, these data suggest that other protein regions must be involved when the attacking nucleophilic group is provided by viral DNA. Because specific recognition of viral DNA ends was previously mapped to the central domain, two different regions of integrase must interact with retroviral DNA.

Need of a site-specific integrating vector in gene therapy has become pressing, as recent work has shown that many of the current integrating vectors used preferentially integrate in the vicinity of genes. A site-specific integrating vector would reduce the risk of insertional mutagenesis posed by r

Need of a site-specific integrating vector in gene therapy has become pressing, as recent work has shown that many of the current integrating vectors used preferentially integrate in the vicinity of genes. A site-specific integrating vector would reduce the risk of insertional mutagenesis posed by randomly integrating vectors, and a non-viral vector would reduce the safety and immunogenicity problems associated with viral vectors. The phiC31 integrase is a protein from Streptomyces phage phiC31 that has been developed as a non-viral site-specific gene therapy vector. The phiC31 integrase catalyzes the integration of a plasmid containing attB into pseudo attP sites in mammalian genomes. It has been shown to function in tissue culture cells as well as in mice. Vectors based on the phiC31 integrase were able to treat tyrosinemia type I in a mouse model and two forms of epidermolysis bullosa in keratinocytes from patients, demonstrating its effectiveness as a gene therapy vector. Development of phiC31 integrase-based vectors is still underway, but it has already been shown to provide long-term expression through site-specific integration.

BACKGROUND: Gene transfer to synovium in joints has been shown to be an effective approach for treating pathologies associated with rheumatoid arthritis (RA) and related joint disorders. However, the efficiency and duration of gene delivery has been limiting for successful gene therapy for arthritis

BACKGROUND: Gene transfer to synovium in joints has been shown to be an effective approach for treating pathologies associated with rheumatoid arthritis (RA) and related joint disorders. However, the efficiency and duration of gene delivery has been limiting for successful gene therapy for arthritis. The transient gene expression that often accompanies non-viral gene delivery can be prolonged by integration of vector DNA into the host genome. We report a novel approach for non-viral gene therapy to joints that utilizes phage phiC31 integrase to bring about unidirectional genomic integration. METHODS: Rabbit and human synovial cells were co-transfected with a plasmid expressing phiC31 integrase and a plasmid containing the transgene and an attB site. Cells were cultured with or without G418 selection and the number of neo-resistant colonies or eGFP cells determined, respectively. Plasmid rescue, PCR query, and DNA sequence analysis were performed to reveal integration sites in the rabbit and human genomes. For in vivo studies, attB-reporter gene plasmids and a plasmid expressing phiC31 integrase were intra-articularly injected into rabbit knees. Joint sections were used for histological analysis of beta-gal expression, and synovial cells were isolated to measure luciferase expression. RESULTS: We demonstrated that co-transfection of a plasmid expressing phiC31 integrase with a plasmid containing the transgene and attB increased the frequency of transgene expression in rabbit synovial fibroblasts and primary human RA synoviocytes. Plasmid rescue and DNA sequence analysis of plasmid-chromosome junctions revealed integration at endogenous pseudo attP sequences in the rabbit genome, and PCR query detected integration at previously characterized integration sites in the human genome. Significantly higher levels of transgene expression were detected in vivo in rabbit knees after intra-articular injection of attB-reporter gene plasmids and a plasmid expressing phiC31 integrase. CONCLUSION: The ability of phiC31 integrase to facilitate genomic integration in synovial cells and increase transgene expression in the rabbit synovium suggests that, in combination with more efficient DNA delivery methods, this integrase system could be beneficial for treatment of rheumatoid arthritis and other joint disorders.

Stem cells can potentially be utilized in combined gene/cell therapies for neural diseases. We examined the ability of the non-viral varphiC31 integrase system to promote stable transgene expression in mouse neural progenitor cells (mNPCs). varphiC31 integrase catalyzes the sequence-specific integra

Stem cells can potentially be utilized in combined gene/cell therapies for neural diseases. We examined the ability of the non-viral varphiC31 integrase system to promote stable transgene expression in mouse neural progenitor cells (mNPCs). varphiC31 integrase catalyzes the sequence-specific integration of attB-containing plasmids into pseudo attP sites in mammalian genomes, to produce long-term transgene expression. We achieved gene transfer by co-nucleofection of a plasmid carrying the luciferase marker gene and an attB site and a plasmid expressing integrase in mNPCs that had been generated in a neurosphere preparation. Luciferase expression was quantified in live cells for 8 weeks, revealing persistence of gene expression. Sequence-specific integration at a preferred pseudo attP site in the mouse genome was detected by using PCR. Furthermore, sustained transgene expression was demonstrated in genetically modified NPCs that were cultured in conditions that promoted either growth or differentiation into neurons and astrocytes. Our results demonstrate that the varphiC31 integrase system produces stable transgene expression in adult mNPCs and their progeny and may be useful in strategies for combating neurodegenerative disorders.

Bacteriophage-encoded serine-integrases are members of the large family of serine-recombinases and catalyze site-specific integrative recombination between a phage attP site and a bacterial attB site to form an integrated prophage. Prophage excision involves a second site-specific recombination even

Bacteriophage-encoded serine-integrases are members of the large family of serine-recombinases and catalyze site-specific integrative recombination between a phage attP site and a bacterial attB site to form an integrated prophage. Prophage excision involves a second site-specific recombination event, in which the sites generated by integration, attL and attR, are used as substrates to regenerate attP and attB. Excision is catalyzed by integrase but also requires a phage-encoded recombination directionality factor (RDF). The Bxb1 recombination sites, attP and attB, are small (<50 bp), different in sequence, and quasisymmetrical, and they give rise to attL- and attR-recombinant products that are asymmetric but similar to each other, each being composed of B- and P-type half-sites. We show here that the determination of correct excision products is a two-step process, with a presynaptic RDF-dependent step that aligns attL and attR in the correct orientation and a postsynaptic step in which the nonpalindromic central dinucleotide confers identity to attL and attR and prevents each from recombining with itself.

The site-specific integrase from bacteriophage phiC31 functions in mammalian cells and is being applied for genetic engineering, including gene therapy. The phiC31 integrase catalyzes precise, unidirectional recombination between its 30-40-bp attP and attB recognition sites. In mammalian cells, the

The site-specific integrase from bacteriophage phiC31 functions in mammalian cells and is being applied for genetic engineering, including gene therapy. The phiC31 integrase catalyzes precise, unidirectional recombination between its 30-40-bp attP and attB recognition sites. In mammalian cells, the enzyme also mediates integration of plasmids bearing attB into native sequences that have partial sequence identity with attP, termed pseudo attP sites. Here, we analyzed the features of phiC31-mediated integration into pseudo attP sites in the human genome. Sequence analysis of 196 independent integration events derived from three cell lines revealed approximately 101 integration sites: 56% of the events were recurrent integrations distributed among 19 pseudo attP sequences. Bioinformatics analysis revealed a approximately 30-bp palindromic consensus sequence motif shared by all of the repeat occurrences and most of the single occurrence sites, verifying that phiC31-mediated integration into pseudo attP sites is significantly guided by DNA sequence recognition. The most favored unique sequence in these cell lines occurred at chromosome 19q13.31 and accounted for 7.5% of integration events. Other frequent integration sites were in three specific sequences in subfamilies of ERVL and L1 repetitive sequences, accounting for an additional 17.9% of integration events. Integrations could occur in either orientation at a pseudo attP site, were often accompanied by small deletions, and typically occurred in a single copy per cell. A number of aberrant events were also described, including large deletions and chromosome rearrangements. phiC31 integrase-mediated integration only slightly favored genes and did not favor promoter regions. Gene density and expression studies suggested chromatin context effects. An analysis of the safety of integration sites in terms of proximity to cancer genes suggested minimal cancer risk. We conclude that integration systems derived from phiC31 integrase have great potential utility.