Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon

Nat Methods. 2009 May;6(5):363-9. doi: 10.1038/nmeth.1323. Epub 2009 Mar 31.

Abstract

Induced pluripotent stem cells (iPSCs) have been generated from somatic cells by transgenic expression of Oct4 (Pou5f1), Sox2, Klf4 and Myc. A major difficulty in the application of this technology for regenerative medicine, however, is the delivery of reprogramming factors. Whereas retroviral transduction increases the risk of tumorigenicity, transient expression methods have considerably lower reprogramming efficiencies. Here we describe an efficient piggyBac transposon-based approach to generate integration-free iPSCs. Transposons carrying 2A peptide-linked reprogramming factors induced reprogramming of mouse embryonic fibroblasts with equivalent efficiencies to retroviral transduction. We removed transposons from these primary iPSCs by re-expressing transposase. Transgene-free iPSCs could be identified by negative selection. piggyBac excised without a footprint, leaving the iPSC genome without any genetic alteration. iPSCs fulfilled all criteria of pluripotency, such as pluripotency gene expression, teratoma formation and contribution to chimeras. piggyBac transposon-based reprogramming may be used to generate therapeutically applicable iPSCs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkaline Phosphatase / metabolism
  • Animals
  • Cell Dedifferentiation / drug effects
  • Cell Dedifferentiation / genetics*
  • Cell Differentiation / physiology
  • Chimera / embryology
  • Chimera / metabolism
  • Chromosomal Proteins, Non-Histone
  • DNA Transposable Elements / genetics*
  • Embryo, Mammalian / cytology
  • Embryo, Mammalian / metabolism
  • Fibroblasts / cytology
  • Fibroblasts / drug effects
  • Gene Expression / genetics
  • Genetic Vectors / genetics
  • Homeodomain Proteins / metabolism
  • Humans
  • Kruppel-Like Transcription Factors / genetics
  • Kruppel-Like Transcription Factors / metabolism
  • Mice
  • Moths / genetics
  • Nanog Homeobox Protein
  • Octamer Transcription Factor-3 / genetics
  • Octamer Transcription Factor-3 / metabolism
  • Pluripotent Stem Cells / cytology*
  • Pluripotent Stem Cells / metabolism
  • Pluripotent Stem Cells / transplantation
  • Proto-Oncogene Proteins c-myc / genetics
  • Proto-Oncogene Proteins c-myc / metabolism
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism
  • Repressor Proteins / genetics
  • SOXB1 Transcription Factors / genetics
  • SOXB1 Transcription Factors / metabolism
  • Teratoma / pathology
  • Transfection
  • Transgenes
  • Transposases / genetics
  • Valproic Acid / pharmacology

Substances

  • Chromosomal Proteins, Non-Histone
  • DNA Transposable Elements
  • Dppa3 protein, mouse
  • GKLF protein
  • Homeodomain Proteins
  • Kruppel-Like Transcription Factors
  • Lin-28 protein, mouse
  • Nanog Homeobox Protein
  • Nanog protein, mouse
  • Octamer Transcription Factor-3
  • Pou5f1 protein, mouse
  • Proto-Oncogene Proteins c-myc
  • RNA-Binding Proteins
  • Repressor Proteins
  • SOXB1 Transcription Factors
  • Sox2 protein, mouse
  • Valproic Acid
  • Transposases
  • Alkaline Phosphatase