An optimized transgenesis system for <i>Drosophila</i> using germ-line-specific φC31 integrases

TLDR

Germ‑line transformation via transposable elements is powerful for studying gene function in *Drosophila*, but transposon‑mediated transgenesis has inherent limitations. The study aims to overcome these limitations by optimizing a phiC31‑based integration system. The authors engineered endogenous phiC31 integrase expression driven by nanos and vasa elements and introduced a white‑gene reconstitution system for visual selection of precise attP targeting, thereby improving transformation efficiency. They produced a library of precisely mapped attP landing sites across the genome, demonstrated in‑situ modification of these sites, and showed that the system offers high efficiency, ease of use, and versatility, enabling systematic high‑throughput screening of large cDNA sets and regulatory elements.

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.