Testing a three-finger zinc finger nuclease using a GFP reporter system.
Homologous recombination is the most precise way to manipulate the genome. It has been used extensively in bacteria, yeast, murine embryonic stem cells, and a few other specialized cell lines, but it has not been available in other systems such as mammalian somatic cells. However, the creation of a gene-specific DNA double-strand break can stimulate homologous recombination by several-thousandfold in mammalian somatic cells. These double-strand breaks can be created in mammalian genomes by zinc finger nucleases (ZFNs), artificial proteins in which a zinc finger DNA-binding domain is fused to a nonspecific nuclease domain. This protocol describes how to test newly designed ZFNs using a cell-based green fluorescent protein (GFP) reporter assay to determine if they are active in a mammalian cell-culture-based system.
Matthew
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Porteus
D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000235:genetics* / Q000378:metabolism*; D005347:Fibroblasts / Q000201:enzymology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D003851:Deoxyribonucleases