Generation of Stable Knockout Mammalian Cells by TALEN-Mediated Locus-Specific Gene Editing

Methods Mol Biol. 2017:1498:107-120. doi: 10.1007/978-1-4939-6472-7_7.

Abstract

Precise and targeted genome editing using Transcription Activator-Like Effector Endonucleases (TALENs) has been widely used and proven to be an extremely effective and specific knockout strategy in both cultured cells and animal models. The current chapter describes a protocol for the construction and generation of TALENs using serial and hierarchical digestion and ligation steps, and using the synthesized TALEN pairs to achieve locus-specific targeted gene editing in mammalian cell lines using a modified clonal selection strategy in an easy and cost-efficient manner.

Keywords: Double-strand break (DSB) repair; Fok1; Gene knockout in mammalian cells; Genome engineering; Homology-directed repair (HDR); Nonhomologous end joining (NHEJ); Repeat variable di-residues (RVDs); Restriction enzyme and ligation (REAL); T7E1 assay; TALEN.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Endonucleases / genetics*
  • Gene Editing / methods
  • Gene Knockout Techniques / methods
  • Genetic Engineering / methods
  • Mammals / genetics*
  • Mice
  • NIH 3T3 Cells
  • RNA Editing
  • Transcription Activator-Like Effector Nucleases / genetics*

Substances

  • Endonucleases
  • Transcription Activator-Like Effector Nucleases