Inducible Transgene Expression in Human iPS Cells Using Versatile All-in-One piggyBac Transposons

Methods Mol Biol. 2016;1357:111-31. doi: 10.1007/7651_2015_251.

Abstract

Transgenics is a mainstay of functional genomics. Conditionally overexpressing genes of interest (GOIs) helps to reveal their roles in the control of complex biological processes. Complemented by findings in classic animal model systems, recent advances in human embryonic stem cell (hESC) and patient-specific induced pluripotent stem cell (hiPSC) differentiation have led to sophisticated in vitro models of human development and disease. Yet, as transgenic elements encoding inducible systems must be introduced de novo into each genetically unique human stem cell line, robust and straightforward solutions to gene delivery are required. Transposons are a family of mobile DNA elements that have been adapted as experimental tools for stable genomic integration of transgenes. The piggyBac (PB) transposon from Trichoplusia ni presents a number of benefits over classic viral or BAC transgenesis: ease of application, simple integration-site mapping, and the unique capacity for traceless excision. Moreover, their large capacity permits the consolidation of multiple transgene components in a single vector system. In this chapter, we outline the features of a panel of "All-in-One" PB transposons designed for drug-inducible gene expression and provide guidelines to establish and validate populations or clones of transgenic hiPSCs.

Keywords: Doxycycline regulated; Gene expression; Human induced pluripotent stem cell (hiPSC); Reprogramming; Stable transgenesis; Transposon; piggyBac.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Culture Techniques / methods*
  • Cell Separation / methods
  • Cell Tracking / methods
  • Cells, Cultured
  • Cellular Reprogramming Techniques / methods*
  • Cellular Reprogramming*
  • Clone Cells / cytology
  • DNA Transposable Elements / genetics*
  • Doxycycline / pharmacology
  • Fibroblasts / cytology
  • Gene Expression / drug effects
  • Gene Expression / genetics*
  • Genes, Reporter
  • Genetic Vectors / genetics*
  • Green Fluorescent Proteins / genetics
  • Humans
  • Induced Pluripotent Stem Cells / cytology*
  • Induced Pluripotent Stem Cells / metabolism
  • Luciferases / genetics
  • Luminescent Proteins / genetics
  • Neomycin / pharmacology
  • Puromycin / pharmacology
  • Transgenes* / genetics

Substances

  • DNA Transposable Elements
  • Luminescent Proteins
  • red fluorescent protein
  • Green Fluorescent Proteins
  • Puromycin
  • Luciferases
  • Neomycin
  • Doxycycline