Design, engineering, and characterization of zinc finger nucleases.
Zinc finger nuclease (ZFN)-mediated gene targeting is rapidly becoming a powerful tool for "gene editing" and "directed mutagenesis" of plant and mammalian genomes including the human genome. ZFN-mediated gene targeting provides molecular biologists with the ability to site-specifically manipulate and permanently modify plant and mammalian genomes. Facile production of ZFNs and rapid characterization of their in vitro sequence-specific cleavage properties are a pre-requisite before ZFN-mediated gene targeting can become an efficient and effective practical tool for widespread use in biotechnology. Here, we report the design, engineering, and rapid in vitro characterization of ZFNs that target specific endogenous sequences within two mouse genes (mTYR and mCFTR), and two human genes (hCCR5 and hDMPK), respectively. These engineered ZFNs recognize their respective cognate DNA sites encoded in a plasmid substrate in a sequence-specific manner and, as expected, they induce a double-strand break at the chosen target site.
Mani
Mala
M
Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205-2179, USA.
Kandavelou
Karthikeyan
K
Dy
Fei Jamie
FJ
Durai
Sundar
S
Chandrasegaran
Srinivasan
S
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D004247:DNA / Q000737:chemistry; D004249:DNA Damage; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics*; D005821:Genetic Techniques; D016678:Genome; D015894:Genome, Human; D018745:Genome, Plant; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D065636:Myotonin-Protein Kinase; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011817:Rabbits; D019713:Receptors, CCR5 / Q000378:metabolism; D011995:Recombination, Genetic; D014158:Transcription, Genetic; D016335:Zinc Fingers
C585517:DMPK protein, human; C585516:DMPK protein, mouse; D019713:Receptors, CCR5; D004247:DNA; D065636:Myotonin-Protein Kinase; D017346:Protein Serine-Threonine Kinases; D004722:Endoribonucleases