A hyperactive piggyBac transposase for mammalian applications

Kosuke Yusa, Liqin Zhou, Meng Amy Li, Allan Bradley, Nancy L. Craig

Research output: Contribution to journalArticle

Abstract

DNA transposons have been widely used for transgenesis and insertional mutagenesis in various organisms. Among the transposons active in mammalian cells, the moth-derived transposon piggyBac is most promising with its highly efficient transposition, large cargo capacity, and precise repair of the donor site. Here we report the generation of a hyperactive piggyBac transposase. The active transposition of piggyBac in multiple organisms allowed us to screen a transposase mutant library in yeast for hyperactive mutants and then to test candidates inmouse ES cells. We isolated 18 hyperactive mutants in yeast, among which five were also hyperactive in mammalian cells. By combining allmutations, a total of 7 aa substitutions, into a single reading frame, we generated a unique hyperactive piggyBac transposase with 17-fold and ninefold increases in excision and integration, respectively. We showed its applicability by demonstrating an increased efficiency of generation of transgene-free mouse induced pluripotent stem cells. We also analyzed whether this hyperactive piggyBac transposase affects the genomic integrity of the host cells. The frequency of footprints left by the hyperactive piggyBac transposase was as low as WT transposase (∼1%) and we found no evidence that the expression of the transposase affects genomic integrity. This hyperactive piggyBac transposase expands the utility of the piggyBac transposon for applications inmammalian genetics and gene therapy.

Original languageEnglish (US)
Pages (from-to)1531-1536
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume108
Issue number4
DOIs
StatePublished - Jan 25 2011

Keywords

  • Gene correction
  • Reprogramming

ASJC Scopus subject areas

  • General

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