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.     

Identification of pseudo attP sites for phage phiC31 integrase in bovine genome

Streptomyces phage phiC31 integrase was found to mediate site-specific integration of foreign genes at pseudo attP sites of genomes in human, mouse, rat, and Drosophila. This paper reports that phiC31 integrase can also mediate homologous recombination between attB and pseudo attP sites in bovine cells and foreign gene integration was increased at least 2-fold in bovine fibroblasts or Madin-Darby bovine kidney (MDBK) cells. Two intrinsic pseudo attP sites named BpsF1 and BpsM1 located in the inter-gene regions on chromosome 28 and 19, respectively, were identified in bovine genome. These pseudo attP sites shared similar characteristics with those from other species as previously described. Our study demonstrated that the phiC31 integrase system provides a new potential for genetic engineering of the bovine genome and might be beneficial for the research on ruminant.     

Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase

We previously established that the phage phiC31 integrase, a site-specific recombinase, mediates efficient integration in the human cell environment at attB and attP phage attachment sites on extrachromosomal vectors. We show here that phage attP sites inserted at various locations in human and mouse chromosomes serve as efficient targets for precise site-specific integration. Moreover, we characterize native "pseudo" attP sites in the human and mouse genomes that also mediate efficient integrase-mediated integration. These sites have partial sequence identity to attP. Such sites form naturally occurring targets for integration. This phage integrase-mediated reaction represents an effective site-specific integration system for higher cells and may be of value in gene therapy and other chromosome engineering strategies.     

A profile of native integration sites used by φC31 integrase in the bovine genome

The Streptomyces phage φC31 integrase can efficiently target attB-bearing transgenes to endogenous pseudo attP sites within mammalian genomes.To better understand the activity ofφC31 integrase in the bovine genome,DNA sequences of 44 integration events were analyzed,and 32 pseudo attP sites were identified.The majority of these sites share a sequence motif that contains inverted repeats and has similarities to wild-type attP site.Genomic DNA flanking these sites typically contained repetitive sequence elements,such as short and long interspersed repetitive elements.These sequence features indicate that DNA sequence recognition plays an important role in guidingφC31-mediated site-specific integration.In addition,BF27 integration hotspot sites were identified in the bovine genome, which accounted for 13.6%of all isolated integration events and mapped to an intron of the deleted in liver cancer 1(DLC1) gene.Also we found that the pseudo attP sites in the bovine genome had other features in common with those in the human genome.This study represents the first time that the sequence features of pseudo attP sites in the bovine genome were analyzed.We conclude that this site-specific integrase system has great potential for applied modifications of the bovine genome.     

The streptomyces genome contains multiple pseudo-attB sites for the (phi)C31-encoded site-specific recombination system

The integrase from the Streptomyces phage (phi)C31 is a member of the serine recombinase family of site-specific recombinases and is fundamentally different from that of lambda or its relatives. Moreover, (phi)C31 int/attP is used widely as an essential component of integration vectors (such as pSET152) employed in the genetic analysis of Streptomyces species. phiC31 or integrating plasmids containing int/attP have been shown previously to integrate at a locus, attB, in the chromosome. The DNA sequences of the attB sites of various Streptomyces species revealed nonconserved positions. In particular, the crossover site was narrowed to the sequence 5'TT present in both attP and attB. Strains of Streptomyces coelicolor and S. lividans were constructed with a deletion of the attB site ((Delta)attB), and pSET152 was introduced into these strains by conjugation. Thus, secondary or pseudo-attB sites were identified by Southern blotting and after rescue of plasmids containing DNA flanking the insertion sites from the chromosome. The sequences of the integration sites had similarity to those of attB. Analysis of the insertions of pSET152 into both attB(+) and (Delta)attB strains indicated that this plasmid can integrate at several loci via independent recombination events within a transconjugant.     

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.     

Phage R4 integrase mediates site-specific integration in human cells.

The R4 integrase is a site-specific, unidirectional recombinase derived from the genome of phage R4 of Streptomyces parvulus. Here we define compact attB and attP recognition sites for the R4 integrase and express the enzyme in mammalian cells. We demonstrate that R4 integrase functions in human cells, performing efficient and precise recombination between R4 attB and attP sites cloned on an extrachromosomal vector. We also provide evidence that the enzyme can mediate integration of an incoming plasmid bearing an attB or attP site into endogenous sequences in the human genome. Furthermore, when R4 attB and attP sites are placed into the human genome, either by random integration or at a specific sequence by using the phi C31 integrase, they act as targets for integration of incoming plasmids bearing R4 att sites. The R4 integrase has immediate utility as a site-specific integration tool for genome engineering, as well as potential for further development.     

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.     

Control of directionality in the site-specific recombination system of the Streptomyces phage phiC31

The genome of the Streptomyces temperate phage phiC31 integrates into the host chromosome via a recombinase belonging to a novel group of phage integrases related to the resolvase/invertase enzymes. Previously, it was demonstrated that, in an in vitro recombination assay, phiC31 integrase catalyses integration (attP/attB recombination) but not excision (attL/attR). The mechanism responsible for this recombination site selectivity was therefore investigated. Purified integrase was shown to bind with similar apparent binding affinities to between 46 bp and 54 bp of DNA at each of the attachment sites, attP, attB, attL and attR. Assays using recombination sites of 50 bp and 51 bp for attP and attB, respectively, showed that these fragments were functional in attP/attB recombination and maintained strict site selectivity, i.e. no recombination between non-permissive sites, such as attP/attP, attB/attL, etc., was observed. Using bandshifts and supershift assays in which permissive and non-permissive combinations of att sites were used in the presence of integrase, only the attP/attB combination could generate supershifts. Recombination products were isolated from the supershifted complexes. It was concluded that these supershifted complexes contained the recombination synapse and that site specificity, and therefore directionality, is determined at the level of stable synapse formation.     

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.     

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.     

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.     

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.     

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.     

φC31整合酶系统介导的位点特异性整合研究进展

来源于链霉菌(Streptomyces)噬菌体φC31的整合酶可介导链霉菌附着位点(attB)和噬菌体附着位点(attP)之间的同源重组,这种重组亦可在多种动植物细胞内进行;而且,该整合酶还可介导含attB位点的载体以位点特异性方式整合于多种真核生物基因组内的假attP位点,并使转基因持续高效表达。因此,φC31整合酶在基因修饰、基因治疗及转基因动物研制等方面得到了广泛的应用。文章就近年来φC31整合酶整合规律、提高效率方面的改进及安全性等相关领域的研究进展进行了综述。     

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.     

Chicken hypersensitive site-4 insulator increases human serum albumin expression in bovine mammary epithelial cells modified with phiC31 integrase

Achieving high expression levels of recombinant human serum albumin (HSA) for purification is a solution for the large amount of plasma-derived HSA needed in therapeutic applications. Here, we employed phiC31 integrase system and chicken hypersensitive site-4 (cHS4) insulators to construct a HSA expression vector for high-level HSA expression. The phiC31 integrase system mediated efficient transgene integration in bovine mammary epithelial cells (bMECs). A preferred pseudo attP site, which had 38 % identity with the 39 bp wild-type attP sequence, was detected in six out of 55 bMEC colonies. Addition of the cHS4 insulator to the phiC31 integrase system resulted in 8–20-fold increases of HSA expression compared with that of using integrase alone. Moreover, the reverse-oriented cHS4 insulator in the phiC31 integrase system provided the optimal level of HSA expression in bMECs.     

Site-Specific Recombination of Temperate Myxococcus xanthus Phage Mx8: Genetic Elements Required for Integration

Like most temperate bacteriophages, phage Mx8 integrates into a preferred locus on the genome of its host, Myxococcus xanthus, by a mechanism of site-specific recombination. The Mx8 int-attP genes required for integration map within a 2.2-kilobase-pair (kb) fragment of the phage genome. When this fragment is subcloned into a plasmid vector, it facilitates the site-specific integration of the plasmid into the 3' ends of either of two tandem tRNAAsp genes, trnD1 and trnD2, located within the attB locus of the M. xanthus genome. Although Int-mediated site-specific recombination occurs between attP and either attB1 (within trnD1) or attB2 (within trnD2), the attP x attB1 reaction is highly favored and often is accompanied by a deletion between attB1 and attB2. The int gene is the only Mx8 gene required in trans for attP x attB recombination. The int promoter lies within the 106-bp region immediately upstream of one of two alternate GTG start codons, GTG-5208 (GTG at bp 5208) and GTG-5085, for integrase and likely is repressed in the prophage state. All but the C-terminal 30 amino acid residues of the Int protein are required for its ability to mediate attP x attB recombination efficiently. The attP core lies within the int coding sequence, and the product of integration is a prophage in which the 3' end of int is replaced by host sequences. The prophage intX gene is predicted to encode an integrase with a different C terminus.     

Site-Specific Recombination of Temperate Myxococcus xanthus Phage Mx8: Regulation of Integrase Activity by Reversible, Covalent Modification

Temperate Myxococcus xanthus phage Mx8 integrates into the attB locus of the M. xanthus genome. The phage attachment site, attP, is required in cis for integration and lies within the int (integrase) coding sequence. Site-specific integration of Mx8 alters the 3' end of int to generate the modified intX gene, which encodes a less active form of integrase with a different C terminus. The phage-encoded (Int) form of integrase promotes attP x attB recombination more efficiently than attR x attB, attL x attB, or attB x attB recombination. The attP and attB sites share a common core. Sequences flanking both sides of the attP core within the int gene are necessary for attP function. This information shows that the directionality of the integration reaction depends on arm sequences flanking both sides of the attP core. Expression of the uoi gene immediately upstream of int inhibits integrative (attP x attB) recombination, supporting the idea that uoi encodes the Mx8 excisionase. Integrase catalyzes a reaction that alters the primary sequence of its gene; the change in the primary amino acid sequence of Mx8 integrase resulting from the reaction that it catalyzes is a novel mechanism by which the reversible, covalent modification of an enzyme is used to regulate its specific activity. The lower specific activity of the prophage-encoded IntX integrase acts to limit excisive site-specific recombination in lysogens carrying a single Mx8 prophage, which are less immune to superinfection than lysogens carrying multiple, tandem prophages. Thus, this mechanism serves to regulate Mx8 site-specific recombination and superinfection immunity coordinately and thereby to preserve the integrity of the lysogenic state.     

PhiC31 integrase-mediated cassette exchange in silkworm embryos

To construct an effective site-specific integration system in the silkworm, we examined if phiC31 integrase works in silkworm embryos. As an assay system, we constructed an extrachromosomal cassette exchange reaction system between two attP sites of an acceptor plasmid and two attB sites of a donor plasmid. To evaluate the activity, integrase mRNAs synthesized from three different plasmids were used. We injected a mixture of the acceptor and donor plasmids with the mRNA synthesized in vitro from one of the three plasmids into silkworm embryos at 4-6?h after oviposition and recovered plasmid DNAs from the embryos 3?days after injection. The resultant plasmids were transformed into Escherichia coli and spread on selection medium plates containing the appropriate antibiotics. A colony-forming assay and restriction enzyme digestion of the plasmids purified from the colonies showed that the phiC31 integrase worked very efficiently in the silkworm embryos. Notably, a phiC31 integrase mRNA synthesized from two of the plasmids produced cassette exchange plasmids at a high frequency, suggesting that the mRNA can be used to construct a targeted integration system in silkworms.