title: Construction and characterization of adenoviral vectors for the delivery of TALENs into human cells.
abstract: Transcription activator-like effector nucleases (TALENs) are designed to cut the genomic DNA at specific chromosomal positions. The resulting DNA double strand break activates cellular repair pathways that can be harnessed for targeted genome modifications. TALENs thus constitute a powerful tool to interrogate the function of DNA sequences within complex genomes. Moreover, their high DNA cleavage activity combined with a low cytotoxicity make them excellent candidates for applications in human gene therapy. Full exploitation of these large and repeat-bearing nucleases in human cell types will benefit largely from using the adenoviral vector (AdV) technology. The genetic stability and the episomal nature of AdV genomes in conjunction with the availability of a large number of AdV serotypes able to transduce various human cell types make it possible to achieve high-level and transient expression of TALENs in numerous target cells, regardless of their mitotic state. Here, we describe a set of protocols detailing the rescue, propagation and purification of TALEN-encoding AdVs. Moreover, we describe procedures for the characterization and quantification of recombinant viral DNA present in the resulting AdV preparations. The protocols are preceded by information about their underlying principles and applied in the context of second-generation capsid-modified AdVs expressing TALENs targeted to the AAVS1 "safe harbor" locus on human chromosome 19.
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands.
authors.forename: Maarten
authors.initials: M
authors.lastname: Holkers
authors.affiliation: Institute for Cell and Gene Therapy, University Medical Center Freiburg, 79106 Freiburg, Germany; Center for Chronic Immunodeficiency, University Medical Center Freiburg, 79106 Freiburg, Germany. Electronic address: toni.cathomen@uniklinik-freiburg.de.
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, 2333 ZC Leiden, The Netherlands. Electronic address: m.goncalves@lumc.nl.
authors.forename: Manuel A F V
authors.initials: MA
authors.lastname: Gonçalves
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D004279:DNA, Viral / Q000008:administration & dosage / Q000235:genetics*; D004720:Endonucleases / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans
chemical_list: D004279:DNA, Viral; D004720:Endonucleases
keywords: Adenoviral vectors; Designer nucleases; Gene editing; Genome engineering; TALENs; Transcription activator-like effector nucleases