Genome Modification of Pluripotent Cells by Using Transcription Activator-Like Effector Nucleases (TALENs)

Methods Mol Biol. 2015:1330:253-67. doi: 10.1007/978-1-4939-2848-4_19.

Abstract

Interest is increasing in transcription activator-like effector nucleases (TALENs) as a tool to introduce targeted double-strand breaks into the large genomes of human and animal cell lines. The produced DNA lesions stimulate DNA repair pathways, error-prone but dominant non-homologous end joining (NHEJ) and accurate but less occurring homology-directed repair (HDR), and as a result targeted genes can be modified. Here, we describe a modified Golden-Gate cloning method for generating TALENs and also details for targeting genes in mouse embryonic stem cells. The protocol described here can be used for modifying the genome of a broad range of pluripotent cell lines.

Keywords: Gene targeting; Golden-Gate cloning; TALENs.

MeSH terms

  • Animals
  • Binding Sites
  • Cloning, Molecular
  • Computational Biology / methods
  • Embryonic Stem Cells
  • Endonucleases / metabolism*
  • Gene Targeting / methods*
  • Genetic Engineering / methods*
  • Genome*
  • Mice
  • Plasmids / genetics
  • Polymerase Chain Reaction
  • Protein Binding
  • Sequence Analysis, DNA
  • Transfection

Substances

  • Endonucleases