Genetics. Published Articles Ahead of Print: February 1, 2006, Copyright © 2006
doi:10.1534/genetics.105.052829


A more recent version of this article appeared on April 1, 2006.

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Efficient Gene Targeting in Drosophila with Zinc Finger Nucleases

Kelly Beumer 1, Gargi Bhattacharyya 2, Marina Bibikova 3, Jonathan K. Trautman 1 and Dana Carroll 4*

1 University of Utah
2 Louisiana State University
3 Illumina, Inc.
4 University of Utah School of Medicine

* To whom correspondence should be addressed. E-mail: dana{at}biochem.utah.edu.

Submitted on October 26, 2005
Revised on January 16, 2006
Accepted on 1 February 2006


Abstract

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 procedure that generates these high frequencies relies on cleavage of the target by designed zinc finger nucleases (ZFNs) and production of a linear donor in situ. Increasing expression of ZFNs led to higher frequencies of gene targeting, demonstrating the beneficial effect of activating the target. In the absence of a homologous donor DNA, ZFN cleavage led to the recovery of new mutants at three loci - y, ry and bw - through nonhomologous end joining (NHEJ) after cleavage. Because zinc fingers can be directed to a broad range of DNA sequences and targeting is very efficient, this approach promises to allow genetic manipulation of many different genes, even in cases where the mutant phenotype cannot be predicted.

Key Words: Drosophila, Gene targeting, homologous recombination, yellow and rosy genes, zinc fingers




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