Combination of Sleeping Beauty transposition and chemically induced dimerization selection for robust production of engineered cells

Nucleic Acids Res. 2012 Jun;40(11):e85. doi: 10.1093/nar/gks213. Epub 2012 Mar 8.

Abstract

The main methods for producing genetically engineered cells use viral vectors for which safety issues and manufacturing costs remain a concern. In addition, selection of desired cells typically relies on the use of cytotoxic drugs with long culture times. Here, we introduce an efficient non-viral approach combining the Sleeping Beauty (SB) Transposon System with selective proliferation of engineered cells by chemically induced dimerization (CID) of growth factor receptors. Minicircles carrying a SB transposon cassette containing a reporter transgene and a gene for the F36VFGFR1 fusion protein were delivered to the hematopoietic cell line Ba/F3. Stably-transduced Ba/F3 cell populations with >98% purity were obtained within 1 week using this positive selection strategy. Copy number analysis by quantitative PCR (qPCR) revealed that CID-selected cells contain on average higher copy numbers of transgenes than flow cytometry-selected cells, demonstrating selective advantage for cells with multiple transposon insertions. A diverse population of cells is present both before and after culture in CID media, although site-specific qPCR of transposon junctions show that population diversity is significantly reduced after selection due to preferential expansion of clones with multiple integration events. This non-viral, positive selection approach is an attractive alternative for producing engineered cells.

Publication types

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

MeSH terms

  • Animals
  • Cell Engineering / methods*
  • Cell Line
  • Cell Line, Transformed
  • Cell Proliferation
  • Cell Survival
  • DNA Transposable Elements*
  • Dimerization
  • Genetic Engineering / methods
  • Green Fluorescent Proteins / genetics
  • Mice
  • Plasmids / genetics
  • Receptor, Fibroblast Growth Factor, Type 1 / genetics
  • Receptor, Fibroblast Growth Factor, Type 1 / metabolism
  • Recombinant Fusion Proteins / metabolism
  • Tacrolimus / analogs & derivatives
  • Tacrolimus / metabolism
  • Tacrolimus Binding Proteins / genetics
  • Tacrolimus Binding Proteins / metabolism
  • Transduction, Genetic
  • Transgenes

Substances

  • AP20187
  • DNA Transposable Elements
  • Recombinant Fusion Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Receptor, Fibroblast Growth Factor, Type 1
  • Tacrolimus Binding Proteins
  • Tacrolimus