Control of directionality in the site‐specific recombination system of the <i>Streptomyces</i> phage φC31

Thorpe, Helena; Wilson, S E; Smith, Margaret Caroline MacHin

doi:10.1046/j.1365-2958.2000.02142.x

Cited by 167 publications

(215 citation statements)

References 42 publications

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“…ϕC31 is a bacteriophage that encodes an integrase that mediates sequence directed recombination between a 34 nucleotide long bacterial attachment site (attB) and a 39 base-pair long phage attachment site (attP). ϕC31 integrase has a high efficiency of recombination, requires no accessory factors 5,6 and has been effectively used to integrate genes into plant cells 7 , mammalian cells 1,[8][9][10][11][12][13] , and Drosophila 14 . The ϕC31 integrase does not require an attP site to have perfect sequence fidelity for it to be recognized and cleaved 9 .…”

Section: Introductionmentioning

confidence: 99%

Transgenic Xenopus laevis embryos can be generated using φC31 integrase

2005

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Phage ϕC31 encodes an integrase that can mediate the insertion of extrachromosomal DNA into genomic DNA 1 . Here we show ϕC31 integrase can be used to generate transgenic Xenopus laevis embryos. mRNA encoding integrase was co-injected with a reporter plasmid containing a CMV promoter driven GFP into one cell embryos. The reporter plasmid was integrated into the genome. GFP expression, though robust, was in a limited number of tissues and varied among the embryos analyzed. We attributed this restriction to transcriptional silencing by chromosome position effects. Modification of the reporter plasmid by bracketing the CMV-GFP region with tandem copies of the chicken β-globin 5′ HS4 insulator 2 relieved position effects. Embryos transgenic with insulated CMV-GFP expressed GFP uniformly. Tissue specific expression was achieved when the CMV promoter was replaced with a 551 base-pair minimal gamma crystallin lens promoter from Xenopus. Embryos transgenic with this plasmid had GFP expression limited to the lens of the eye. We observed that about a third of embryos assayed one week after fertilization were transgenic. These experiments demonstrate that the integration of insulated gene sequences using ϕC31 integrase can be used to efficiently create transgenic embryos in Xenopus laevis and may increase the practical use of ϕC31 integrase in other systems as well.The ability to generate transgenic animals has revolutionized studies on gene function. Transgenic frogs of the genus Xenopus have been made using methods based on a restriction-enzyme mediated sperm integration approach described by Kroll and Amaya 3 . This method has been remarkably successful, but can be technically demanding. In addition, many embryos contain multiple copies of the transgene inserted at random insertion sites 3, 4 .We have tried an alternative approach, creating transgenic Xenopus embryos by using ϕC31 integrase mediated recombination. ϕC31 is a bacteriophage that encodes an integrase that mediates sequence directed recombination between a 34 nucleotide long bacterial attachment site (attB) and a 39 base-pair long phage attachment site (attP). ϕC31 integrase has a high efficiency of recombination, requires no accessory factors 5,6 and has been effectively used to integrate genes into plant cells 7 , mammalian cells 1,[8][9][10][11][12][13] , and Drosophila 14 . The ϕC31 integrase does not require an attP site to have perfect sequence fidelity for it to be recognized and cleaved 9 . These imperfect attP sites, or psuedo-attP sites, may have a similarity as low as 24% to an attP site and still allow recombination 9 . It has been estimated that a mammalian genome may contain 100-1000 psuedo attP sites 9 . Since the Xenopus laevis genome is approximately the same size as a mammalian genome (3×10 9 base pairs), we hypothesized that there would also be psuedo-attP sites in its genome that could be used to create transgenic Xenopus embryos. In the experiments that follow all the figure 1e. The half life of the GFP protein has been estimate...

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Section: Introductionmentioning

confidence: 99%

Transgenic Xenopus laevis embryos can be generated using φC31 integrase

2005

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“…The bacteriophage C31 encodes a serine integrase that mediates sequence-directed recombination between a bacterial attachment site (attB) and a phage attachment site (attP) (13). Apart from the site-specificity, another feature of the C31 system is that the integrase solely mediates integration (13), which distinguishes it from most other commonly used systems, such as Cre/loxP or FLP/FRT, where the recombinase can catalyze both the integration and the excision reactions.…”

mentioning

confidence: 99%

An optimized transgenesis system for Drosophila using germ-line-specific φC31 integrases

Bischof

Maeda²,

Hediger³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

1,792

2,089

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Germ-line transformation via transposable elements is a powerful tool to study gene function in Drosophila melanogaster. However, some inherent characteristics of transposon-mediated transgenesis limit its use for transgene analysis. Here, we circumvent these limitations by optimizing a C31-based integration system. We generated a collection of lines with precisely mapped attP sites that allow the insertion of transgenes into many different predetermined intergenic locations throughout the fly genome. By using regulatory elements of the nanos and vasa genes, we established endogenous sources of the C31 integrase, eliminating the difficulties of coinjecting integrase mRNA and raising the transformation efficiency. Moreover, to discriminate between specific and rare nonspecific integration events, a white gene-based reconstitution system was generated that enables visual selection for precise attP targeting. Finally, we demonstrate that our chromosomal attP sites can be modified in situ, extending their scope while retaining their properties as landing sites. The efficiency, ease-of-use, and versatility obtained here with the C31-based integration system represents an important advance in transgenesis and opens up the possibility of systematic, highthroughput screening of large cDNA sets and regulatory elements.attP landing sites ͉ germ-line transformation ͉ site-specific integration A major goal in the present era of genomics is to identify and functionally characterize all genes relevant to a specific pathway or biological process. With its powerful repertoire of genetic tools, the multicellular model organism Drosophila melanogaster has played an eminent role in this endeavor (1). One method to identify relevant genes is to perform chemical mutagenesis screens of various kinds. Another very fruitful approach in Drosophila has been the use of P-element-mediated germ-line transformation (2, 3), especially when combined with tools such as the Gal4/UAS expression system (4) or when it is used for insertional mutagenesis (5, 6). One characteristic of P-elements is their random integration behavior. Although this ''randomness'' is advantageous for generating mutations and deletions, it is generally not ideal for transgene analysis. The random integration of P-elements necessitates considerable effort to map insertions. Genomic position effects complicate the analysis of transgenes and render precise structure/ function analyses nearly impossible. A further shortcoming of the P-element system is its relatively moderate transformation efficiency, a significant hurdle to any large-scale transgenesis effort.Strategies have been developed to circumvent the problem of randomness by targeted integration systems in Drosophila, which are generally based on the FLP and Cre recombinases (7-9, 32). Such techniques permit precise targeting to genomic landing sites but are still handicapped by transformation rates that are, at best, moderately higher than those achieved with the P-element system (8). Furthermore, especially for FL...

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“…One of the resulting lethality lines, #67, was shown to cause 100% conditional embryonic lethality and males of this strain were highly competitive relative to wild-type medfly males in laboratory and field cage tests (6); (ii) the strain carrying insertion 1260 F-3 m-1 has been evaluated to potentially improve monitoring of pest management programs by fluorescent sperm marking and showed no disadvantages in preliminary laboratory competitiveness assays (7). Both (i) and (ii) transgene insertions are single transposon integrations as proven by Southern blots and inverse PCR (6, 7) and therefore contain a single 52-bp attP recombination site that can serve as the landing site for attB-containing plasmids (20).…”

Section: Development and Test Of Piggybac Jumpstarter Strains In Medflymentioning

confidence: 99%

“…For integrase-mediated germline transformation two plasmids containing a 51-bp attB recombination site (20) and an additional 3Ј piggyBac ITR were generated: pSLaf 3ЈpBac-attB PUb-DsRed af (#1252) and pSLaf 3ЈpBac-Ͼ-attB PUb-EGFP af (#1255) carrying a red fluorescent (DsRed) or a green fluorescent marker (EGFP) under the control of the PUb promoter (21,22), respectively ( Fig. 3).…”

Section: Development and Test Of Piggybac Jumpstarter Strains In Medflymentioning

confidence: 99%

Site-specific recombination for the modification of transgenic strains of the Mediterranean fruit fly Ceratitis capitata

Schetelig

Scolari

Handler

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Insect transgenesis is mainly based on the random genomic integration of DNA fragments embedded into non-autonomous transposable elements. Once a random insertion into a specific location of the genome has been identified as particularly useful with respect to transgene expression, the ability to make the insertion homozygous, and lack of fitness costs, it may be advantageous to use that location for further modification. Here we describe an efficient method for the modification of previously inserted transgenes by the use of the site-specific integration system from phage phiC31 in a tephritid pest species, the Mediterranean fruit fly Ceratitis capitata. First, suitable transgenic strains with randomly integrated attP landing sites within transposon-based vectors were identified by molecular and functional characterization. Second, donor plasmids containing an attB site, with additional markers, and transposon ends were integrated into attP sites by phiC31 integrase-mediated recombination. Third, transposase-encoding 'jumpstarter' strains were created and mated to transgenic strains resulting in the postintegrational excision of transposon ends, which left stably integrated transgene insertions that could not be remobilized. This three-step integration and stabilization system will allow the combination of several transgeneencoded advantageous traits at evaluated genomic positions to generate optimized strains for pest control that minimize environmental concerns.insect pest management ͉ phiC31 integrase ͉ transgene stability

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Control of directionality in the site‐specific recombination system of the Streptomyces phage φC31

Cited by 167 publications

References 42 publications

Transgenic Xenopus laevis embryos can be generated using φC31 integrase

Transgenic Xenopus laevis embryos can be generated using φC31 integrase

An optimized transgenesis system for Drosophila using germ-line-specific φC31 integrases

Site-specific recombination for the modification of transgenic strains of the Mediterranean fruit fly Ceratitis capitata

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