title: Zinc-finger nucleases-based genome engineering to generate isogenic human cell lines.
abstract: Customized zinc-finger nucleases (ZFNs) have developed into a promising technology to precisely alter mammalian genomes for biomedical research, biotechnology, or human gene therapy. In the context of synthetic biology, the targeted integration of a transgene or reporter cassette into a "neutral site" of the human genome, such as the AAVS1 locus, permits the generation of isogenic human cell lines with two major advantages over standard genetic manipulation techniques: minimal integration site-dependent effects on the transgene and, vice versa, no functional perturbation of the host-cell transcriptome. Here we describe in detail how ZFNs can be employed to target integration of a transgene cassette into the AAVS1 locus and how to characterize the targeted cells by PCR-based genotyping.
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Anne-Kathrin
authors.initials: AK
authors.lastname: Dreyer
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003851:Deoxyribonucleases / Q000737:chemistry* / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods*; D056426:Genetic Loci / Q000235:genetics; D016678:Genome / Q000235:genetics*; D060005:Genotyping Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D014162:Transfection; D016335:Zinc Fingers
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D003851:Deoxyribonucleases