Chimeric piggyBac transposases for genomic targeting in human cells

Jesse B. Owens, Johann Urschitz, Ilko Stoytchev, Nong C. Dang, Zoia Stoytcheva, Mahdi Belcaid, Kommineni J. Maragathavally, Craig J. Coates, David Segal, Stefan Moisyadi

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

Integrating vectors such as viruses and transposons insert transgenes semi-randomly and can potentially disrupt or deregulate genes. For these techniques to be of therapeutic value, a method for controlling the precise location of insertion is required. The piggyBac (PB) transposase is an efficient gene transfer vector active in a variety of cell types and proven to be amenable to modification. Here we present the design and validation of chimeric PB proteins fused to the Gal4 DNA binding domain with the ability to target transgenes to pre-determined sites. Upstream activating sequence (UAS) Gal4 recognition sites harbored on recipient plasmids were preferentially targeted by the chimeric Gal4-PB transposase in human cells. To analyze the ability of these PB fusion proteins to target chromosomal locations, UAS sites were randomly integrated throughout the genome using the Sleeping Beauty transposon. Both N- and C-terminal Gal4-PB fusion proteins but not native PB were capable of targeting transposition nearby these introduced sites. A genome-wide integration analysis revealed the ability of our fusion constructs to bias 24 of integrations near endogenous Gal4 recognition sequences. This work provides a powerful approach to enhance the properties of the PB system for applications such as genetic engineering and gene therapy.

Original languageEnglish (US)
Pages (from-to)6978-6991
Number of pages14
JournalNucleic Acids Research
Volume40
Issue number14
DOIs
StatePublished - Aug 2012

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Transposases
Aptitude
Transgenes
Genetic Therapy
Genome
Beauty
Proteins
Genetic Engineering
Genes
Plasmids
Viruses
DNA
Recognition (Psychology)
Therapeutics

ASJC Scopus subject areas

  • Genetics

Cite this

Owens, J. B., Urschitz, J., Stoytchev, I., Dang, N. C., Stoytcheva, Z., Belcaid, M., ... Moisyadi, S. (2012). Chimeric piggyBac transposases for genomic targeting in human cells. Nucleic Acids Research, 40(14), 6978-6991. https://doi.org/10.1093/nar/gks309

Chimeric piggyBac transposases for genomic targeting in human cells. / Owens, Jesse B.; Urschitz, Johann; Stoytchev, Ilko; Dang, Nong C.; Stoytcheva, Zoia; Belcaid, Mahdi; Maragathavally, Kommineni J.; Coates, Craig J.; Segal, David; Moisyadi, Stefan.

In: Nucleic Acids Research, Vol. 40, No. 14, 08.2012, p. 6978-6991.

Research output: Contribution to journalArticle

Owens, JB, Urschitz, J, Stoytchev, I, Dang, NC, Stoytcheva, Z, Belcaid, M, Maragathavally, KJ, Coates, CJ, Segal, D & Moisyadi, S 2012, 'Chimeric piggyBac transposases for genomic targeting in human cells', Nucleic Acids Research, vol. 40, no. 14, pp. 6978-6991. https://doi.org/10.1093/nar/gks309
Owens JB, Urschitz J, Stoytchev I, Dang NC, Stoytcheva Z, Belcaid M et al. Chimeric piggyBac transposases for genomic targeting in human cells. Nucleic Acids Research. 2012 Aug;40(14):6978-6991. https://doi.org/10.1093/nar/gks309
Owens, Jesse B. ; Urschitz, Johann ; Stoytchev, Ilko ; Dang, Nong C. ; Stoytcheva, Zoia ; Belcaid, Mahdi ; Maragathavally, Kommineni J. ; Coates, Craig J. ; Segal, David ; Moisyadi, Stefan. / Chimeric piggyBac transposases for genomic targeting in human cells. In: Nucleic Acids Research. 2012 ; Vol. 40, No. 14. pp. 6978-6991.
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