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Published Online November 30, 2006
Science DOI: 10.1126/science.1134426

Research Articles

Submitted on August 28, 2006
Accepted on November 9, 2006

P[acman]: A BAC Transgenic Platform for Targeted Insertion of Large DNA Fragments in D. melanogaster

Koen J. T. Venken 1, Yuchun He 2, Roger A. Hoskins 3, Hugo J. Bellen 4*

1 Program in Developmental Biology
2 Department of Molecular and Human Genetics; Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, USA.
3 Department of Genome Biology, Lawrence Berkeley National Laboratory, Berkeley, CA 94720-3200, USA.
4 Program in Developmental Biology; Department of Molecular and Human Genetics; Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, USA; Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA.

* To whom correspondence should be addressed.
Hugo J. Bellen , E-mail: hbellen{at}bcm.edu

We describe a transgenesis platform for Drosophila melanogaster which integrates three recently-developed technologies: a conditionally amplifiable bacterial artificial chromosome (BAC), recombineering, and {phi}C31-mediated transgenesis. The BAC is maintained at low-copy number, facilitating plasmid maintenance and recombineering, but induced to high-copy number for plasmid isolation. Recombineering allows gap repair and mutagenesis in bacteria. Gap repair efficiently retrieves DNA fragments up to 133 kb from P1 or BAC clones. {phi}C31-mediated transgenesis integrates these large DNA fragments at specific sites in the genome, allowing rescue of lethal mutations in the corresponding genes. This transgenesis platform should greatly facilitate structure-function analyses of most Drosophila genes.


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