title: Generation of Stable Knockout Mammalian Cells by TALEN-Mediated Locus-Specific Gene Editing.
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.
authors.affiliation: P1/12 CIT Scheme VIIM, Division of Molecular Medicine, Bose Institute, Kolkata, 700054, India.
authors.forename: Barun
authors.initials: B
authors.lastname: Mahata
authors.affiliation: P1/12 CIT Scheme VIIM, Division of Molecular Medicine, Bose Institute, Kolkata, 700054, India. kbiswas_1@yahoo.com.
authors.forename: Kaushik
authors.initials: K
authors.lastname: Biswas
mesh_terms: D000818:Animals; D002460:Cell Line; D004720:Endonucleases / Q000235:genetics*; D000072669:Gene Editing / Q000379:methods; D055786:Gene Knockout Techniques / Q000379:methods; D005818:Genetic Engineering / Q000379:methods; D008322:Mammals / Q000235:genetics*; D051379:Mice; D041681:NIH 3T3 Cells; D017393:RNA Editing; D000069896:Transcription Activator-Like Effector Nucleases / Q000235:genetics*
chemical_list: D004720:Endonucleases; D000069896:Transcription Activator-Like Effector Nucleases
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