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Versatile P[acman] BAC libraries for transgenesis studies in Drosophila melanogaster

Abstract

We constructed Drosophila melanogaster bacterial artificial chromosome libraries with 21-kilobase and 83-kilobase inserts in the P[acman] system. We mapped clones representing 12-fold coverage and encompassing more than 95% of annotated genes onto the reference genome. These clones can be integrated into predetermined attP sites in the genome using ΦC31 integrase to rescue mutations. They can be modified through recombineering, for example, to incorporate protein tags and assess expression patterns.

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Figure 1: The P[acman] BAC vector and mapped clones in the eve region.
Figure 2: Expression of EGFP fusion proteins in transgenic embryos.

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Acknowledgements

We thank members of the Washington University Genome Sequencing Center for their excellent BAC end-sequencing services. We thank members of the Bloomington Drosophila Stock Center for providing flies, members of US National Cancer Institute (NCI) Frederick for recombineering reagents, A. Hyman (Max Planck Institute, Dresden) for the LAP-tag plasmid, R. Karess (Centre National de la Recherche Scientifique) for flies, R. Ordway (Penn State University) for flies, J. Reinitz (Stony Brook University) for antibodies, D. Schmucker (Harvard Medical School) for flies, T. Schwarz (Children's Hospital, Boston) for flies, B. Wakimoto (University of Washington) for flies, L. Zipursky (University of California Los Angeles) for flies, and J. Bischof, K. Basler (University of Zurich) and F. Karch (University of Geneva) for providing germline FC31 sources and information about their use. We thank J. Cohen for help with recombineering, N. Giagtzoglou and A. Rajan for help with microscopy, C. Amemiya and D. Frisch for helpful communications and discussions, and B. Wakimoto for critical reading of the manuscript. Confocal microscopy was supported by the Baylor College of Medicine Intellectual and Developmental Disabilities Research Center. This work was supported by a grant from the Howard Hughes Medical Institute to H.J.B. and the US National Institutes of Health modENCODE project in collaboration with K.P.W.

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Correspondence to Hugo J Bellen.

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Supplementary Figures 1–6, Supplementary Tables 1–5, Supplementary Note, Supplementary Discussion (PDF 482 kb)

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Venken, K., Carlson, J., Schulze, K. et al. Versatile P[acman] BAC libraries for transgenesis studies in Drosophila melanogaster. Nat Methods 6, 431–434 (2009). https://doi.org/10.1038/nmeth.1331

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