title: Genome Modification of Pluripotent Cells by Using Transcription Activator-Like Effector Nucleases (TALENs).
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.
authors.affiliation: Department of Animal Science, Ferdowsi University of Mashhad, Mashhad, Iran. taheri1362@gmail.com.
authors.forename: Amir
authors.initials: A
authors.lastname: Taheri-Ghahfarokhi
authors.affiliation: Monash Institute for Medical Research, Monash University, Clayton, VIC, 3168, Australia.|Australian Animal Health Laboratories, CSIRO Biosecurity Flagship, East Geelong, VIC, 3219, Australia.
authors.forename: Luis F
authors.initials: LF
authors.lastname: Malaver-Ortega
authors.affiliation: Department of Chemistry and Biotechnology, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, VIC, 3122, Australia.
authors.forename: Huseyin
authors.initials: H
authors.lastname: Sumer
mesh_terms: D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D019295:Computational Biology / Q000379:methods; D053595:Embryonic Stem Cells; D004720:Endonucleases / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D016678:Genome; D051379:Mice; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017422:Sequence Analysis, DNA; D014162:Transfection
chemical_list: D004720:Endonucleases
keywords: Gene targeting; Golden-Gate cloning; TALENs