Article

Site-directed transposon integration in human cells.

Department of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA 94305-5208, USA.
Nucleic Acids Research (Impact Factor: 8.81). 02/2007; 35(7):e50. DOI: 10.1093/nar/gkm089
Source: PubMed

ABSTRACT The Sleeping Beauty (SB) transposon is a promising gene transfer vector that integrates nonspecifically into host cell genomes. Herein, we attempt to direct transposon integration into predetermined DNA sites by coupling a site-specific DNA-binding domain (DBD) to the SB transposase. We engineered fusion proteins comprised of a hyperactive SB transposase (HSB5) joined via a variable-length linker to either end of the polydactyl zinc-finger protein E2C, which binds a unique sequence on human chromosome 17. Although DBD linkage to the C-terminus of SB abolished activity in a human cell transposition assay, the N-terminal addition of the E2C or Gal4 DBD did not. Molecular analyses indicated that these DBD-SB fusion proteins retained DNA-binding specificity for their respective substrate molecules and were capable of mediating bona fide transposition reactions. We also characterized transposon integrations in the presence of the E2C-SB fusion protein to determine its potential to target predefined DNA sites. Our results indicate that fusion protein-mediated tethering can effectively redirect transposon insertion site selection in human cells, but suggest that stable docking of integration complexes may also partially interfere with the cut-and-paste mechanism. These findings illustrate the feasibility of directed transposon integration and highlight potential means for future development.

0 Bookmarks
 · 
123 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Gene vectors derived from DNA transposable elements have become powerful molecular tools in biomedical research and are slowly moving into the clinic as carriers of therapeutic genes. Conventional uses of DNA transposon-based gene vehicles rely on the intracellular production of the transposase protein from transfected nucleic acids. The transposase mediates mobilization of the DNA transposon, which is typically provided in the context of plasmid DNA. In recent work, we established lentiviral protein transduction from Gag precursors as a new strategy for direct delivery of the transposase protein. Inspired by the natural properties of infecting viruses to carry their own enzymes, we loaded lentivirus-derived particles not only with vector genomes carrying the DNA transposon vector but also with hundreds of transposase subunits. Such particles were found to drive efficient transposition of the piggyBac transposable element in a range of different cell types, including primary cells, and offer a new transposase delivery approach that guarantees short-term activity and limits potential cytotoxicity. DNA transposon vectors, originally developed and launched as a non-viral alternative to viral integrating vectors, have truly become viral. Here, we briefly review our findings and speculate on the perspectives and potential advantages of transposase delivery by lentiviral protein transduction.
    Mobile genetic elements. 01/2014; 4:e29591.
  • [Show abstract] [Hide abstract]
    ABSTRACT: The ability to manipulate the genomes of many insects has become a practical reality over the past 15 years. This has been led by the identification of several useful transposon vector systems that have allowed the identification and development of generalized, species-specific, and tissue-specific promoter systems for controlled expression of gene products upon introduction into insect genomes. Armed with these capabilities, researchers have made significant strides in both fundamental and applied transgenics in key model systems such as Bombyx mori, Tribolium casteneum, Aedes aegypti, and Anopheles stephensi. Limitations of transposon systems were identified, and alternative tools were developed, thus significantly increasing the potential for applied transgenics for control of both agricultural and medical insect pests. The next 10 years promise to be an exciting time of transitioning from the laboratory to the field, from basic research to applied control, during which the full potential of gene manipulation in insect systems will ultimately be realized.
    Annual Review of Entomology 01/2012; 57:267-89. · 13.59 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The discovery of biological concepts can often provide a framework for the development of novel molecular tools, which can help us to further understand and manipulate life. One recent example is the elucidation of the prokaryotic adaptive immune system, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) that protects bacteria and archaea against viruses or conjugative plasmids. The immunity is based on small RNA molecules that are incorporated into versatile multi-domain proteins or protein complexes and specifically target viral nucleic acids via base complementarity. CRISPR/Cas interference machines are utilized to develop novel genome editing tools for different organisms. Here, we will review the latest progress in the elucidation and application of prokaryotic CRISPR/Cas systems and discuss possible future approaches to exploit the potential of these interference machineries.
    International Journal of Molecular Sciences 01/2013; 14(7):14518-31. · 2.46 Impact Factor

Full-text (2 Sources)

View
49 Downloads
Available from
May 22, 2014