Therapeutic applications of the ΦC31 integrase system.

Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120, USA.
Current Gene Therapy (Impact Factor: 4.91). 08/2011; 11(5):375-81.
Source: PubMed

ABSTRACT The potential use of the ΦC31 integrase system in gene therapy opens up the possibilities of new treatments for old diseases. ΦC31 integrase mediates the integration of plasmid DNA into the chromsomes of mammalian cells in a sequence-specific manner, resulting in robust, long-term transgene expression. In this article, we review how ΦC31 integrase mediates transgene integration into the genomes of target cells and summarize the recent preclinical applications of the system to gene therapy. These applications encompass in vivo studies in liver and lung, as well as increasing ex vivo uses of the system, including in neural and muscle stem cells, in cord-lining epithelial cells, and for the production of induced pluripotent stem cells. The safety of the ΦC31 integrase system for gene therapy is evaluated, and its ability to provide treatments for hemophilia is discussed. We conclude that gene therapy strategies utilizing ΦC31 integrase offer great promise for the development of treatments in the future.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Mammalian cells can be reprogrammed into induced pluripotent stem cells (iPSCs), a valuable tool for in vitro disease modeling and regenerative medicine. These applications demand for iPSCs devoid of reprogramming factor transgenes, but current procedures for the derivation of transgene-free iPSCs are inefficient and cumbersome. Here, we describe a new approach for the simple derivation of transgene-free iPSCs by the sequential use of two DNA recombinases, C31 Integrase and Cre, to control the genomic insertion and excision of a single, non-viral reprogramming vector. We show that such transgene-free iPSCs exhibit gene expression profiles and pluripotent developmental potential comparable to genuine, blastocyst-derived embryonic stem cells. As shown by a reporter iPSC line for the differentiation into midbrain dopaminergic neurons, the dual recombinase approach offers a simple and efficient way to derive transgene-free iPSCs for studying disease mechanisms and cell replacement therapies.
    Molecular Biotechnology 03/2014; · 2.28 Impact Factor
  • Source
    Christopher D Krause, Lara S Izotova, Sidney Pestka
    [Show abstract] [Hide abstract]
    ABSTRACT: A bewildering variety of plasmids have been developed for a wide array of purposes in biotechnology and biomedical research. These purposes include transient and stable protein expression, mutation, recombination-mediated nuclear gene knockout, promoter analysis, RNA stability, targeted RNA degradation, and gene therapy. Each plasmid has been optimized for only one of these functions, and consequentially, convenient restriction enzyme sites surround only one genetic element. However, an optimal expression system often requires the use of different genetic elements that are rarely found on the same plasmid (such as differing promoters or eukaryotic antibiotic resistance), and exchange of these elements between plasmids is difficult. We retrofitted pcDNA3 (an archaic but well-validated and useful plasmid from Stratagene, Inc.) to facilitate the exchange of various genetic elements by inserting useful restriction endonuclease sites at the borders of these elements. With the retrofitted plasmid, called pc3.5, we can exchange not only the ectopically expressed gene of interest but also promoters or polyadenylation sites of the gene of interest, as well as the eukaryotic antibiotic resistance gene, its promoter or polyadenylation sequences. We have used derivatives of these plasmids for transient and stable gene expression, promoter analysis and induction, and mesenchymal stem cell engineering. Retrofitting plasmids is a simple process than can be applied to a wide variety of plasmid or viral vector systems to diversify and optimize their utility.
    Advances in Genetics Research, Volume 13, Edited by Kevin V. Urbano, 01/2014: chapter 1: pages 1-22; Nova Publishers., ISBN: 978-1-63463-091-7
  • [Show abstract] [Hide abstract]
    ABSTRACT: The Streptomyces bacteriophage, φC31, uses a site-specific integrase enzyme to perform efficient recombination. The recombination system uses specific sequences to integrate exogenous DNA from the phage into a host. The sequences are known as the attP site in the phage and the attB site in the host. The system can be used as a genetic manipulation tool. In this study it has been applied to the transformation of cultured BmN cells and the construction of transgenic Bombyx mori individuals. A plasmid, pSK-attB/Pie1-EGFP/Zeo-PASV40, containing a cassette designed to express a egfp-zeocin fusion gene, was co-transfected into cultured BmN cells with a helper plasmid, pSK-Pie1/NLS-Int/NSL. Expression of the egfp-zeocin fusion gene was driven by an ie-1 promoter, downstream of a φC31 attB site. The helper plasmid encoded the φC31 integrase enzyme, which was flanked by two nuclear localization signals. Expression of the egfp-zeocin fusion gene could be observed in transformed cells. The two plasmids were also transferred into silkworm eggs to obtain transgenic silkworms. Successful integration of the fusion gene was indicated by the detection of green fluorescence, which was emitted by the silkworms. Nucleotide sequence analysis demonstrated that the attB site had been cut, to allow recombination between the attB and endogenous pseudo attP sites in the cultured silkworm cells and silkworm individuals.
    Molecular Biology Reports 07/2014; 41(10). · 1.96 Impact Factor


Available from
Jun 2, 2014