A phage integrase directs efficient site-specific integration in human cells

Groth, Amy C.; Olivares, Eric C.; Thyagarajan, Bhaskar; Calos, Michèle P.

doi:10.1073/pnas.090527097

Cited by 464 publications

(458 citation statements)

References 21 publications

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“…A 230 bp SpeI fragment containing the fC31 attP site from pTA-attP (Groth et al, 2000) was cloned into the SpeI site of pSL(PhHsp70a-DsRed) to generate pSL(attP;PhHsp70a-DsRed). The attP-PhHsp70a-DsRed construct was cloned as an AscI fragment into the Minos vectors pMi{3xP3-DsRed} and pMi{3xP3-EGFP} (Pavlopoulos and Averof, 2005;Pavlopoulos et al, 2004), generating pMi{3xP3-DsRed;attP;PhHsp70a-DsRed} and pMi{3xP3-EGFP;attP;PhHsp70a-DsRed}.…”

Section: Gene-trapping Constructsmentioning

confidence: 99%

“…Plasmid pBS(MiL;attB;PhHS-EGFP) was generated as follows: we removed the right inverted repeat of Minos from pMi{3xP3-DsRed} by AvrII and NheI digestion and religation; we excised DsRed with SgrBI and NotI and replaced it with a SgrBI-NotI fragment of pSL(PhHS-EGFP) carrying PhHS-EGFP; we then excised 3xP3 using SalI and replaced it with a SalI fragment containing attB from plasmid pTA-attB (Groth et al, 2000).…”

Section: Itrac Constructsmentioning

confidence: 99%

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A versatile strategy for gene trapping and trap conversion in emerging model organisms

et al. 2011

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SUMMARYGenetic model organisms such as Drosophila, C. elegans and the mouse provide formidable tools for studying mechanisms of development, physiology and behaviour. Established models alone, however, allow us to survey only a tiny fraction of the morphological and functional diversity present in the animal kingdom. Here, we present iTRAC, a versatile gene-trapping approach that combines the implementation of unbiased genetic screens with the generation of sophisticated genetic tools both in established and emerging model organisms. The approach utilises an exon-trapping transposon vector that carries an integrase docking site, allowing the targeted integration of new constructs into trapped loci. We provide proof of principle for iTRAC in the emerging model crustacean Parhyale hawaiensis: we generate traps that allow specific developmental and physiological processes to be visualised in unparalleled detail, we show that trapped genes can be easily cloned from an unsequenced genome, and we demonstrate targeting of new constructs into a trapped locus. Using this approach, gene traps can serve as platforms for generating diverse reporters, drivers for tissue-specific expression, gene knockdown and other genetic tools not yet imagined.

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Section: Gene-trapping Constructsmentioning

confidence: 99%

Section: Itrac Constructsmentioning

confidence: 99%

A versatile strategy for gene trapping and trap conversion in emerging model organisms

et al. 2011

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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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“…GUTLESS Ad GENOME 27 When FC31 excises attB/attPflanked sequences, it creates new sequences called attR/ attL preventing the opposite reaction. Thus, the helper adenovirus carrying an attB/attP-flanked packaging signal opens the door to a new gutless production system (authors' unpublished results).…”

Section: Helper Admentioning

confidence: 99%

Gutless adenovirus: last-generation adenovirus for gene therapy

2005

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Last-generation adenovirus vectors, also called helper-dependent or gutless adenovirus, are very attractive for gene therapy because the associated in vivo immune response is highly reduced compared to first-and second-generation adenovirus vectors, while maintaining high transduction efficiency and tropism. Nowadays, gutless adenovirus is administered in different organs, such as the liver, muscle or the central nervous system achieving high-level and long-term transgene expression in rodents and primates. However, as devoid of all viral coding regions, gutless vectors require viral proteins supplied in trans by a helper virus. To remove contamination by a helper virus from the final preparation, different systems based on the excision of the helper-packaging signal have been generated. Among them, Cre-loxP system is mostly used, although contamination levels still are 0.1-1% too high to be used in clinical trials. Recently developed strategies to avoid/reduce helper contamination were reviewed. Gene Therapy (2005) 12, S18-S27.

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A phage integrase directs efficient site-specific integration in human cells

Cited by 464 publications

References 21 publications

A versatile strategy for gene trapping and trap conversion in emerging model organisms

A versatile strategy for gene trapping and trap conversion in emerging model organisms

Transgenic Xenopus laevis embryos can be generated using φC31 integrase

Gutless adenovirus: last-generation adenovirus for gene therapy

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