phiC31-integrase-mediated, site-specific integration of transgenes in the silkworm, Bombyx mori (Lepidoptera: Bombycidae)

Transgenic silkworms can be useful for investigating the functions of genes in the post-genomic era. However, the common method of using a transposon as an insertion tool may result in the random integration of a foreign gene into the genome and suffer from a strong position effect. To overcome these problems, it is necessary to develop a site-specific integration system. It is known that phiC31 integrase has the capacity to mediate recombination between the target sequences attP and attB. To test the availability of site-specific integration in the silkworm, we first examined the efficiency of recombination between the target sites of the two plasmids in silkworm embryos and found that the frequency of recombination was very high. Then we constructed a host strain that possessed the target sequence attP using the common method. We injected the donor plasmid together with the phiC31 integrase mRNA into the embryos of the host strain and obtained positive lines. Structural analysis of the lines showed that site-specific integration occurred by recombination between the genomic attP site and the attB site of the donor plasmid. We can conclude from the results that phiC31 integrase has the ability to mediate the site-specific integration of transgenes into the silkworm chromosome.     

Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31

The phiC31 integrase functions efficiently in vitro and in Escherichia coli, yeast, and mammalian cells, mediating unidirectional site-specific recombination between its attB and attP recognition sites. Here we show that this site-specific integration system also functions efficiently in Drosophila melanogaster in cultured cells and in embryos. Intramolecular recombination in S2 cells on transfected plasmid DNA carrying the attB and attP recognition sites occurred at a frequency of 47%. In addition, several endogenous pseudo attP sites were identified in the fly genome that were recognized by the integrase and used as substrates for integration in S2 cells. Two lines of Drosophila were created by integrating an attP site into the genome with a P element. phiC31 integrase injected into embryos as mRNA functioned to promote integration of an attB-containing plasmid into the attP site, resulting in up to 55% of fertile adults producing transgenic offspring. A total of 100% of these progeny carried a precise integration event at the genomic attP site. These experiments demonstrate the potential for precise genetic engineering of the Drosophila genome with the phiC31 integrase system and will likely benefit research in Drosophila and other insects.     

PhiC31 integrase mediates integration in cultured synovial cells and enhances gene expression in rabbit joints

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.     

An inducible constitutive expression system in Bombyx mori mediated by phiC31 integrase

Inducible gene-expression systems play important roles in gene functional assays in the post-genome era. Streptomyces phage-derived phiC31 integrase, which mediates an irreversible site-specific cassette exchange between the phage attachment site (attP) and the bacterial attachment site (attB), provides a promising option for the construction of a controllable gene-expression system. Here, we report a phiC31 integrase-mediated promoter flip system (FLIP) for the inducible expression of target genes in silkworm (Bombyx mori). First, we constructed a FLIP reporter system, in which a BmAct4 promoter with enhanced translational efficiency was flanked by the attB and attP sites in a head-to-head orientation and further linked in a reverse orientation to a DsRed reporter gene. The coexpression of a C-terminal modified phiC31-NLS integrase carrying a simian virus 40 (SV40) nuclear localization signal (NLS) effectively flipped the BmAct4 promoter through an attB/attP exchange, thereby activating the downstream expression of DsRed in a silkworm embryo-derived cell line, BmE. Subsequently, the FLIP system, together with a system continuously expressing the phiC31-NLS integrase, was used to construct binary transgenic silkworm lines. Hybridization between FLIP and phiC31-NLS transgenic silkworm lines resulted in the successful flipping of the BmAct4 promoter, with an approximately 39% heritable transformation efficiency in silkworm offspring, leading to the constitutive and high-level expression of DsRed in silkworms, which accounted for approximately 0.81% of the silkworm pupal weight. Our successful development of the FLIP system offers an effective alternative for manipulating gene expression in silkworms and other lepidopteran species.     

Using phiC31 integrase to make transgenic Xenopus laevis embryos

Bacteriophage phiC31 produces the enzyme integrase that allows the insertion of the phage genome into its bacterial host. This enzyme recognizes a specific DNA sequence in the phage (attP) and a different sequence in the bacterium (attB). Recombination between these sites leads to integration in a reaction that requires no accessory factors. Seminal studies by the Calos laboratory demonstrated that the phiC31 integrase was capable of integrating plasmid with an attB site into mammalian genomes at sites that approximated the attP site. We describe the use of attB-containing plasmids with insulated reporter genes for the successful integration of DNA into Xenopus embryos. The method offers a way to produce transgenic embryos without manipulation of sperm nuclei using microinjection methods that are standard for experiments in Xenopus laevis. The method aims to allow the non-mosaic controlled expression of new genetic material in the injected embryo and compares favorably with the time that is normally taken to analyze embryos injected with mRNAs, plasmids, morpholinos or oligonucleotides.     

Transgenic Xenopus laevis embryos can be generated using phiC31 integrase

Bacteriophage phiC31 encodes an integrase that can mediate the insertion of extrachromosomal DNA into genomic DNA. Here we show that the coinjection of mRNA encoding phiC31 integrase with plasmid DNA encoding the green fluorescent protein (GFP) can be used to generate transgenic X. laevis embryos. Despite integration into the genome, appropriate promoter expression required modification of the reporter plasmid by bracketing the GFP reporter gene with tandem copies of the chicken beta-globin 5' HS4 insulator to relieve silencing owing to chromatin position effects. These experiments demonstrate that the integration of insulated gene sequences using phiC31 integrase can be used to efficiently create transgenic embryos in X. laevis and may increase the practical use of phiC31 integrase in other systems as well.     

In vivo site-specific integration of transgene in silkworm via PhiC31 integrase-mediated cassette exchange

Current techniques for genetic engineering of the silkworm Bombyx mori genome utilize transposable elements, which result in positional effects and insertional mutagenesis through random insertion of exogenous DNA. New methods for introducing transgenes at specific positions are therefore needed to overcome the limitations of transposon-based strategies. Although site-specific recombination systems have proven powerful tools for genome manipulation in many organisms, their use has not yet been well established for the integration of transgenes in the silkworm. We describe a method for integrating target genes at pre-defined chromosomal sites in the silkworm via phiC31/att site-specific recombination system-mediated cassette exchange. Successful recombinase-mediated cassette exchange (RMCE) was observed in the two transgenic target strains with an estimated transformation efficiency of 3.84–7.01%. Our results suggest that RMCE events between chromosomal attP/attP target sites and incoming attB/attB sites were more frequent than those in the reciprocal direction. This is the first report of in vivo RMCE via phiC31 integrase in the silkworm, and thus represents a key step toward establishing genome manipulation technologies in silkworms and other lepidopteran species.     

Transgene excision in zebrafish using the phiC31 integrase

Genome engineering strategies employing site‐specific recombinases (SSRs) have become invaluable to the study of gene function in model organisms. One such SSR, the integrase encoded by the Streptomyces bacteriophage phiC31, promotes recombination between heterotypic attP and attB sites. In the present study I have examined the feasibility of the use of phiC31 integrase for intramolecular recombination strategies in zebrafish embryos. I report here that (1) phiC31 integrase is functional in zebrafish cells, (2) phiC31 integrase can excise a transgene cassette flanked by an attB and an attP site, analogous to a common use of the Cre/lox SSR system, (3) phiC31 integrase functions in the zebrafish germline, and (4) a phiC31 integrase‐estrogen receptor hormone‐binding domain variant fusion protein catalyzes attB‐attP recombination in zebrafish embryos in a 4‐hydroxytamoxifen‐dependent manner, albeit less efficiently than phiC31 alone. These features should make this a useful approach for genome manipulations in the zebrafish. genesis 48:137–143, 2010. © 2010 Wiley‐Liss, Inc.     

Adjusting the attB site in donor plasmid improves the efficiency of ΦC31 integrase system

ΦC31 integrase, a site-specific recombinase, can catalyze integration of circular DNA bearing attB site into pseudo attP sites in mammalian genomes. However, the integration efficiency mediated by integrase is relatively low. Our study centered on the investigation of the impact of the position, orientation, and number of attBs in the donor plasmid on the efficiency of ΦC31 integrase system. Donor plasmids bearing various types of attBs (including forward and reverse directions, tandem, and intersperse) and reporter enhanced green fluorescent protein (EGFP) were constructed. The plasmids plus helper plasmid encoding integrase were co-transfected into HeLa cells. After G418 selection, the resistant cell colonies were counted for calculating chromosomal integration frequency. EGFP expression was detected by fluorescence-activated cell sorter and enzyme-linked immunosorbent assay analysis. The results showed that efficiency of integration mediated by integrase accounted for 70% ± 7.1% of total integration events in the transfected HeLa cells. Compared with a forward orientation of attB in donor plasmid, a reverse direction of attB or interspersed attBs showed 1.5- or 2.8-fold increase in integration efficiency, respectively, while tandem attBs in donor plasmids caused a decreased efficiency of integration. We conclude that the adjustment of attB sites in donor plasmids may be of value for gene therapy and routine genetic engineering by using ΦC31 integrase system.     

Site-specific genomic integration produces therapeutic Factor IX levels in mice

We used the integrase from phage phiC31 to integrate the human Factor IX (hFIX) gene permanently into specific sites in the mouse genome. A plasmid containing attB and an expression cassette for hFIX was delivered to the livers of mice by using high-pressure tail vein injection. When an integrase expression plasmid was co-injected, hFIX serum levels increased more than tenfold to approximately 4 microg/ml, similar to normal FIX levels, and remained stable throughout the more than eight months of the experiment. hFIX levels persisted after partial hepatectomy, suggesting genomic integration of the vector. Site-specific integration was proven by characterizing and quantifying genomic integration in the liver at the DNA level. Integration was documented at two pseudo-attP sites, native sequences with partial identity to attP, with one site highly predominant. This study demonstrates in vivo gene transfer in an animal by site-specific genomic integration.     

Site-specific cassette exchange and germline transmission with mouse ES cells expressing phiC31 integrase

Currently two site-specific recombinases are available for engineering the mouse genome: Cre from P1 phage and Flp from yeast. Both enzymes catalyze recombination between two 34-base pair recognition sites, lox and FRT, respectively, resulting in excision, inversion, or translocation of DNA sequences depending upon the location and the orientation of the recognition sites. Furthermore, strategies have been designed to achieve site-specific insertion or cassette exchange. The problem with both recombinase systems is that when they insert a circular DNA into the genome (trans event), two cis-positioned recognition sites are created, which are immediate substrates for excision. To stabilize the trans event, functional mutant recognition sites had to be identified. None of the systems, however, allowed efficient selection-free identification of insertion or cassette exchange. Recently, an integrase from Streptomyces phage phiC31 has been shown to function in Schizosaccharomyces pombe and mammalian cells. This enzyme recombines between two heterotypic sites: attB and attP. The product sites of the recombination event (attL and attR) are not substrates for the integrase. Therefore, the phiC31 integrase is ideal to facilitate site-specific insertions into the mammalian genome.     

Creating transgenic Drosophila by microinjecting the site-specific phiC31 integrase mRNA and a transgene-containing donor plasmid

We describe a microinjection-based phiC31 integrase mRNA-mediated method for creating transgenic Drosophila strains. This approach is more efficient than traditional methods and ensures that the transgene is targeted to a precise genomic position. The method involves targeting integration of an exogenous plasmid (containing the transgene and sequences to facilitate integration) to a preplaced recipient site in the Drosophila genome. The plasmid is coinjected into embryos with mRNA encoding the phiC31 integrase, the enzyme that catalyzes the integration reaction. Using the protocol described here, transgenic lines can be established from, on average, 46% of fertile adults obtained after injection, and all integrations should be targeted to the chosen genomic insertion site. The whole procedure, from injection to established transgenic stocks, can be completed in three generations (approximately 1 month) and can be adapted for other types of transgenesis and mRNA injections in Drosophila.     

Integration specificity of phage phiC31 integrase in the human genome

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.     

Switching the polarity of a bacteriophage integration system

During lysogenic growth many temperate bacteriophage genomes are integrated into the host's chromosome and efficient integration and excision are therefore an essential part of the phage life cycle. The Streptomyces phage phiC31 encodes an integrase related to the resolvase/invertases and is evolutionarily and mechanistically distinct from the integrase of phage lambda. We show that during phiC31 integration the polarity of the recombination sites, attB and attP, is dependent on the sequences of the two base pairs (bp) where crossover occurs. A loss or switch in polarity of the recombination sites can occur by mutation of this dinucleotide, leading to incorrectly joined products. The properties of the mutant sites implies that phiC31 integrase interacts symmetrically with the substrates, which during synapsis can align apparently freely in either of two alternative forms that lead to correct or incorrect joining of products. Analysis of the topologies of the reaction products provided evidence that integrase can synapse and activate strand exchange even when recombinant products cannot form due to mismatches at the crossover site. The topologies of the recombination products are complex and indicative of multiple pathways to product formation. The efficiency of integration of a phiC31 derivative, KC859, into an attB site with switched polarity was assayed in vivo and shown to be no different from integration into a wild-type attB. Thus neither the host nor KC859 express a factor that influences the alignment of the recombination sites at synapsis.     

Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage phiC31 site-specific recombination system

The site-specific recombination system used by the Streptomyces bacteriophage phiC31 was tested in the fission yeast Schizosaccharomyces pombe. A target strain with the phage attachment site attP inserted at the leu1 locus was co-transformed with one plasmid containing the bacterial attachment site attB linked to a ura4+ marker, and a second plasmid expressing the phiC31 integrase gene. High-efficiency transformation to the Ura+ phenotype occurred when the integrase gene was expressed. Southern analysis revealed that the attB-ura4+ plasmid integrated into the chromosomal attP site. Sequence analysis showed that the attBxattP recombination was precise. In another approach, DNA with a ura4+ marker flanked by two attB sites in direct orientation was used to transform S. pombe cells bearing an attP duplication. The phiC31 integrase catalyzed two reciprocal cross-overs, resulting in a precise gene replacement. The site-specific insertions are stable, as no excision (the reverse reaction) was observed on maintenance of the integrase gene in the integrant lines. The irreversibility of the phiC31 site-specific recombination system sets it apart from other systems currently used in eukaryotic cells, which reverse readily. Deployment of the phiC31 recombination provides new opportunities for directing transgene and chromosome rearrangements in eukaryotic systems.     

Phage phiC31 integrase-mediated genomic integration of the common cytokine receptor gamma chain in human T-cell lines

BACKGROUND: X-linked severe combined immunodeficiency (SCID-X1, X-SCID) is a life-threatening disease caused by a mutated common cytokine receptor gamma chain (gammac) gene. Although ex vivo gene therapy, i.e., transduction of the gammac gene into autologous CD34(+) cells, has been successful for treating SCID-X1, the retrovirus vector-mediated transfer allowed dysregulated integration, causing leukemias. Here, to explore an alternative gene transfer methodology that may offer less risk of insertional mutagenesis, we employed the phiC31 integrase-based integration system using human T-cell lines, including the gammac-deficient ED40515(-). METHODS: A phiC31 integrase and a neo(r) gene expression plasmid containing the phiC31 attB sequence were co-delivered by electroporation into Jurkat cells. After G418 selection, integration site analyses were performed using linear amplification mediated-polymerase chain reaction (LAM-PCR). ED40515(-) cells were also transfected with a gammac expression plasmid containing attB, and the integration sites were determined. IL-2 stimulation was used to assess the functionality of the transduced gammac in an ED40515(-)-derived clone. RESULTS: Following co-introduction of the phiC31 integrase expression plasmid and the plasmid carrying attB, the efficiency of integration into the unmodified human genome was assessed. Several integration sites were characterized, including new integration sites in intergenic regions on chromosomes 13 and 18 that may be preferred in hematopoietic cells. An ED40515(-) line bearing the integrated gammac gene exhibited stable expression of the gammac protein, with normal IL-2 signaling, as assessed by STAT5 activation. CONCLUSIONS: This study supports the possible future use of this phiC31 integrase-mediated genomic integration strategy as an alternative gene therapy approach for treating SCID-X1.     

PhiC31 integrase induces efficient site-specific excision in zebrafish

Site-specific recombinases catalyze recombination between specific targeting sites to delete, insert, invert, or exchange DNA with high fidelity. In addition to the widely used Cre and Flp recombinases, the phiC31 integrase system from Streptomyces phage may also be used for these genetic manipulations in eukaryotic cells. Unlike Cre and Flp, phiC31 recognizes two heterotypic sites, attB and attP, for recombination. While the phiC31 system has been recently applied in mouse and human cell lines and in Drosophila, it has not been demonstrated whether it can also catalyze efficient DNA recombination in zebrafish. Here we show that phiC31 integrase efficiently induces site-specific deletion of episomal targets as well as chromosomal targets in zebrafish embryos. Thus, the phiC31 system can be used in zebrafish for genetic manipulations, expanding the repertoire of available tools for genetic manipulation in this vertebrate model.     

Phage phiC31 integrase-mediated genomic integration and long-term gene expression in the lung after nonviral gene delivery

BACKGROUND: Phage phiC31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study investigated the activity of phiC31 integrase in murine lungs. METHODS: Transfections in murine alveolar epithelial (MLE12) cells were performed with Lipofectamine 2000. For in vivo gene delivery, DNA was complexed with polyethylenimine (PEI) and PEI-DNA complexes were injected intravenously into mice. Expression of luciferase in mice was monitored by in vivo bioluminsecence imaging. Genomic integration and integration into a previously described 'hotspot' were confirmed by polymerase chain reaction (PCR). RESULTS: phiC31 integrase mediated intramolecular recombination between wild-type attB and attP sites in MLE12 cells. Long-term gene expression could be observed in MLE12 cells in the presence of integrase without any selection pressure. Long-term expression of luciferase after intravenous injection of PEI-DNA complexes could be observed only in the lungs of mice which were co-injected with the integrase-encoding plasmid. Increased amounts of integrase plasmid and administration of a second dose had no effect on the level of luciferase expression achieved with a single dose, which was three orders of magnitude lower than the values observed on 'day 1' post application. Genomic integration of the transgene in the mouse lungs was confirmed by PCR. Seven out of the fifteen treated mice showed integration at the mpsL1 site, a previously described 'hot spot' from liver. CONCLUSIONS: These results provide evidence for the activity of phiC31 integrase in lungs but also emphasize the need for optimization of the system to maintain long-term gene expression at high levels.     

Site-specific transformation of Drosophila via phiC31 integrase-mediated cassette exchange

Position effects can complicate transgene analyses. This is especially true when comparing transgenes that have inserted randomly into different genomic positions and are therefore subject to varying position effects. Here, we introduce a method for the precise targeting of transgenic constructs to predetermined genomic sites in Drosophila using the C31 integrase system in conjunction with recombinase-mediated cassette exchange (RMCE). We demonstrate the feasibility of this system using two donor cassettes, one carrying the yellow gene and the other carrying GFP. At all four genomic sites tested, we observed exchange of donor cassettes with an integrated target cassette carrying the mini-white gene. Furthermore, because RMCE-mediated integration of the donor cassette is necessarily accompanied by loss of the target cassette, we were able to identify integrants simply by the loss of mini-white eye color. Importantly, this feature of the technology will permit integration of unmarked constructs into Drosophila, even those lacking functional genes. Thus, C31 integrase-mediated RMCE should greatly facilitate transgene analysis as well as permit new experimental designs.     

Targeted engineering of the Drosophila genome

The application of phiC31 phage integrase in Drosophila for unidirectional and site-specific DNA integration was pioneered by Groth et al. in 2004 1 and quickly triggered a wave of innovative tools taking advantage of these unique properties of phiC31. Three recent papers have further developed novel approaches that combine the phiC31-mediated DNA integration with the homologous recombination (HR)-based gene targeting 2 3 for the purpose of efficient and targeted modifications of Drosophila genomic loci. Despite significant differences, the general strategies are similar in principle in the SIRT (site-specific integrase mediated repeated targeting) approach by Gao et al. 4, the IMAGO (integrase-mediated approach for gene knock-out) approach by Choi et al. 5 and the genomic engineering approach developed by our group 6. All three use HR-based gene targeting to first implant a single or a pair of phiC31-attP recombination sites into the target locus. Flies carrying such targeted insertions of attP sites can then be used as "founder lines", in which modified DNA sequences ("knock-in DNA") can be repeatedly and efficiently inserted back into the target locus via phiC31-mediated integration. Thus, by carrying out the targeting experiments only once, one can then directedly and efficiently modify the target locus into virtually any desired knock-in allele. Here we give a brief overview of the SIRT, IMAGO, and genomic engineering approaches and propose a revised genomic engineering scheme in which a single ends-out targeting event will generate founder lines suitable for both recombinase-mediated cassette exchange (RMCE) and single-site based integration of knock-in DNA.