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An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases.

Proceedings of the National Academy of Sciences of the United States of America

Animals, Attachment Sites, Microbiological, genetics, Base Sequence, Computational Biology, Cytogenetic Analysis, Drosophila melanogaster, Gene Targeting, methods, Gene Transfer Techniques, Integrases, Molecular Sequence Data, Polymerase Chain Reaction, Sequence Analysis, DNA, Transformation, Genetic, Transgenes, Virus Integration

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      Abstract

      Germ-line transformation via transposable elements is a powerful tool to study gene function in Drosophila melanogaster. However, some inherent characteristics of transposon-mediated transgenesis limit its use for transgene analysis. Here, we circumvent these limitations by optimizing a phiC31-based integration system. We generated a collection of lines with precisely mapped attP sites that allow the insertion of transgenes into many different predetermined intergenic locations throughout the fly genome. By using regulatory elements of the nanos and vasa genes, we established endogenous sources of the phiC31 integrase, eliminating the difficulties of coinjecting integrase mRNA and raising the transformation efficiency. Moreover, to discriminate between specific and rare nonspecific integration events, a white gene-based reconstitution system was generated that enables visual selection for precise attP targeting. Finally, we demonstrate that our chromosomal attP sites can be modified in situ, extending their scope while retaining their properties as landing sites. The efficiency, ease-of-use, and versatility obtained here with the phiC31-based integration system represents an important advance in transgenesis and opens up the possibility of systematic, high-throughput screening of large cDNA sets and regulatory elements.

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      Author and article information

      Journal
      17360644
      1805588
      10.1073/pnas.0611511104

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