Jonathan K. Trautman

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We report very high gene targeting frequencies in Drosophila by direct embryo injection of mRNAs encoding specific zinc-finger nucleases (ZFNs). Both local mutagenesis via nonhomologous end joining (NHEJ) and targeted gene replacement via homologous recombination (HR) have been achieved in up to 10% of all targets at a given locus. In embryos that are wild(More)
Chimeric nucleases that are hybrids between a nonspecific DNA cleavage domain and a zinc finger DNA recognition domain were tested for their ability to find and cleave their target sites in living cells. Both engineered DNA substrates and the nucleases were injected into Xenopus laevis oocyte nuclei, in which DNA cleavage and subsequent homologous(More)
Gene targeting—the process of gene replacement by homologous recombination—is a very useful but typically inefficient technique (1). A general method for improving the efficiency of gene targeting would be valuable in many circumstances, as would the extension of this genetic tool to a broader range of organisms. Model experiments have demonstrated that a(More)
This report describes high-frequency germline gene targeting at two genomic loci in Drosophila melanogaster, y and ry. In the best case, nearly all induced parents produced mutant progeny; 25% of their offspring were new mutants and most of these were targeted gene replacements resulting from homologous recombination (HR) with a marked donor DNA. The(More)
In near-field scanning optical microscopy, a light source or detector with dimensions less than the wavelength (lambda) is placed in close proximity (lambda/50) to a sample to generate images with resolution better than the diffraction limit. A near-field probe has been developed that yields a resolution of approximately 12 nm ( approximately lambda/43) and(More)
The near-field optical interaction between a sharp probe and a sample of interest can be exploited to image, spectroscopically probe, or modify surfaces at a resolution (down to approximately 12 nm) inaccessible by traditional far-field techniques. Many of the attractive features of conventional optics are retained, including noninvasiveness, reliability,(More)
Gene targeting is the term commonly applied to experimental gene replacement by homologous recombination (HR). This process is substantially stimulated by a double-strand break (DSB) in the genomic target. Zinc-finger nucleases (ZFNs) are targetable cleavage reagents that provide an effective means of introducing such a break in conjunction with delivery of(More)
The B800-to-B850 energy transfer time in the purified B800-850 light-harvesting complex of Rhodobacter sphaeroides 2.4.1 is determined to be 0.7 ps at room temperature. The electronic state dynamics of the principal carotenoid of this species, spheroidene, are examined, both in vivo and in vitro, by direct femtosecond time-resolved experiments and by(More)
Using zinc-finger nucleases (ZFNs) to cleave the chromosomal target, we have achieved high frequencies of gene targeting in the Drosophila germline. Both local mutagenesis through nonhomologous end joining (NHEJ) and gene replacement via homologous recombination (HR) are stimulated by target cleavage. In this study we investigated the mechanisms that(More)
cDNAs for the Xenopus laevis homologue of the endo/exonuclease FEN-1 (DNase IV) have been cloned using a polymerase chain reaction strategy. Products were obtained from two nonallelic Xenopus genes (xFEN-1a and xFEN-1b) that differ from each other by 4.5% in amino acid sequence. Both are 80% identical to mammalian FEN-1 proteins and 55% identical to the(More)