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A toolkit for high-throughput, cross-species gene engineering in Drosophila

Abstract

We generated two complementary genomic fosmid libraries for Drosophila melanogaster and Drosophila pseudoobscura that permit seamless modification of large genomic clones by high-throughput recombineering and direct transgenesis. The fosmid transgenes recapitulated endogenous gene expression patterns. These libraries, in combination with recombineering technology, will be useful to rescue mutant phenotypes, allow imaging of gene products in living flies and enable systematic analysis and manipulation of gene activity across species.

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Figure 1: Genomic fosmid libraries.
Figure 2: Tagged fosmid clones recapitulate wild-type gene expression patterns.

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Acknowledgements

We thank Z. Maliga, E. Knust and C. Bökel for critical reading of the manuscript, S. Preibisch and D. White for three-dimensional reconstructions of multiview images, and P. Mejstrik for technical help. R.K.E. was supported by Dresden International Graduate School for Biomedicine and Bioengineering PhD stipend.

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Authors and Affiliations

Authors

Contributions

R.K.E. did the majority of the work presented in this manuscript. M.S. performed the high-throughput recombineering experiments. S.W. set up the fosmid sequencing protocol. K.A.L. generated the D. pseudoobscura library. P.T. conceived the whole project and wrote the manuscript.

Corresponding author

Correspondence to Pavel Tomancak.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Tables 1–4, Supplementary Protocols 1–3 (PDF 1125 kb)

Supplementary Video 1

3D rendering of an embryo expressing FlyFos (CG4702-GFP) imaged by SPIM. (MOV 1441 kb)

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Ejsmont, R., Sarov, M., Winkler, S. et al. A toolkit for high-throughput, cross-species gene engineering in Drosophila. Nat Methods 6, 435–437 (2009). https://doi.org/10.1038/nmeth.1334

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