A nucleolus-predominant piggyBac transposase, NP-mPB, mediates elevated transposition efficiency in mammalian cells

Jin Bon Hong, Fu Ju Chou, Amy T. Ku, Hsiang Hsuan Fan, Tung Lung Lee, Yung Hsin Huang, Tsung Lin Yang, I. Chang Su, I. Shing Yu, Shu Wha Lin, Chung Liang Chien, Hong Nerng Ho, You Tzung Chen

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2 Citations (Scopus)

Abstract

PiggyBac is a prevalent transposon system used to deliver transgenes and functionally explore the mammalian untouched genomic territory. The important features of piggyBac transposon are the relatively low insertion site preference and the ability of seamless removal from genome, which allow its potential uses in functional genomics and regenerative medicine. Efforts to increase its transposition efficiency in mammals were made through engineering the corresponding transposase (PBase) codon usage to enhance its expression level and through screening for mutant PBase variants with increased enzyme activity. To improve the safety for its potential use in regenerative medicine applications, site-specific transposition was achieved by using engineered zinc finger- and Gal4-fused PBases. An excision-prone PBase variant has also been successfully developed. Here we describe the construction of a nucleolus-predominant PBase, NP-mPB, by adding a nucleolus-predominant (NP) signal peptide from HIV-1 TAT protein to a mammalian codon-optimized PBase (mPB). Although there is a predominant fraction of the NP-mPB-tGFP fusion proteins concentrated in the nucleoli, an insertion site preference toward nucleolar organizer regions is not detected. Instead a 3-4 fold increase in piggyBac transposition efficiency is reproducibly observed in mouse and human cells.

Original languageEnglish
Article numbere89396
JournalPLoS ONE
Volume9
Issue number2
DOIs
Publication statusPublished - Feb 24 2014
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

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    Hong, J. B., Chou, F. J., Ku, A. T., Fan, H. H., Lee, T. L., Huang, Y. H., Yang, T. L., Su, I. C., Yu, I. S., Lin, S. W., Chien, C. L., Ho, H. N., & Chen, Y. T. (2014). A nucleolus-predominant piggyBac transposase, NP-mPB, mediates elevated transposition efficiency in mammalian cells. PLoS ONE, 9(2), [e89396]. https://doi.org/10.1371/journal.pone.0089396