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- W1975384911 abstract "In the backwards world of genetics, the aberrant function of a mutant gene often reveals its normal role in the life of the individual. The whole spectrum of mutations (altered activity, overexpression, dominant-negative) has been brought to bear in this quest, but null mutations (those that yield no product) occupy a place of special importance: they set the baseline for all comparisons. So critical is this reference point that researchers in yeast have created deletion libraries in which every known gene and suspicious ORF has been surgically removed, from start codon to stop codon, using the scalpel of homologous recombination (HR). Although this stem-to-stern knockout strategy is not practical in most eukaryotes, HR has been used routinely, if tediously, to eliminate gene function in mammalian cells. There, the main barrier to facile gene knockout is not the low frequency of HR, as often stated, but rather the overwhelming preponderance of homology-independent integrations that obscure the targeted event (1). In this issue of PNAS, Santiago et al. (2) have turned this standard approach on its head: forsaking HR in favor of nonhomologous end joining (NHEJ). They exploit a custom-designed zinc-finger nuclease (ZFN) to cleave the dihydrofolate reductase (DHFR) gene, and then search for cells that have inaccurately repaired the lesion by NHEJ. Remarkably, >1% of the treated cells were found to have both DHFR alleles modified in a way that completely abolished their activity. This work extends to mammalian cells the observation that ZFNs can induce gene-specific, NHEJ-based mutations at high frequency in flies (3, 4), worms (5), and plants (6)." @default.
- W1975384911 created "2016-06-24" @default.
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- W1975384911 date "2008-04-15" @default.
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- W1975384911 title "Knockout punches with a fistful of zinc fingers" @default.
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- W1975384911 doi "https://doi.org/10.1073/pnas.0802298105" @default.
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