An efficient and reversible transposable system for gene delivery and lineage-specific differentiation in human embryonic stem cells

Cell Stem Cell. 2009 Sep 4;5(3):332-42. doi: 10.1016/j.stem.2009.07.011.


Unraveling the therapeutic potential of human embryonic stem cells (hESC) requires tools to modify their genome. We have engineered the PiggyBac transposable element to create an efficient system for gene delivery in hESCs. This redesigned system, named "ePiggyBac," can deliver up to 18 Kb inserts, and transgene expression is observed in almost 90% of hES cells. ePiggyBac transposons can also carry insulators, inducible expression cassettes, and short hairpin RNAs for gain- and loss-of-function approaches. In hES cells, ePiggyBac's efficiency is superior to that of viral vectors and previously described transposons, including other PiggyBac-based systems. In addition, ePiggyBac transgenes can be removed from the hESC genome without leaving any mutation. We used this system to direct hESC differentiation toward a neuronal phenotype. We then removed the transposons to obtain transgene-free neuronal precursors and neurons. The ability to create fully reversible genetic modifications represents an important step toward clinical applications of hESCs.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Cell Differentiation* / drug effects
  • Cell Lineage* / drug effects
  • DNA Transposable Elements / genetics*
  • Doxycycline / pharmacology
  • Embryonic Stem Cells / cytology*
  • Embryonic Stem Cells / drug effects
  • Embryonic Stem Cells / metabolism*
  • Gene Transfer Techniques*
  • Genetic Engineering
  • Genome, Human / genetics
  • Humans
  • Macaca
  • Molecular Sequence Data
  • Mutagenesis, Insertional / drug effects
  • Neurons / cytology
  • Neurons / drug effects
  • Neurons / metabolism
  • Organ Specificity / drug effects
  • Phenotype
  • RNA, Small Interfering / metabolism
  • Transgenes


  • DNA Transposable Elements
  • RNA, Small Interfering
  • Doxycycline