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Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes

Abstract

A major obstacle to creating precisely expressed transgenes lies in the epigenetic effects of the host chromatin that surrounds them. Here we present a strategy to overcome this problem, employing a Gal4-inducible luciferase assay to systematically quantify position effects of host chromatin and the ability of insulators to counteract these effects at phiC31 integration loci randomly distributed throughout the Drosophila genome. We identify loci that can be exploited to deliver precise doses of transgene expression to specific tissues. Moreover, we uncover a previously unrecognized property of the gypsy retrovirus insulator to boost gene expression to levels severalfold greater than at most or possibly all un-insulated loci, in every tissue tested. These findings provide the first opportunity to create a battery of transgenes that can be reliably expressed at high levels in virtually any tissue by integration at a single locus, and conversely, to engineer a controlled phenotypic allelic series by exploiting several loci. The generality of our approach makes it adaptable to other model systems to identify and modify loci for optimal transgene expression.

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Figure 1: The UAS::luciferase reporter before and after integration at attP docking sites.
Figure 2: Levels of basal and inducible expression at attP landing sites are uncorrelated.
Figure 3: Position effects are tissue dependent.
Figure 4: Exploiting position effects to create an allelic series.
Figure 5: The gypsy insulator increases Gal4-inducible gene expression in larval and adult tissues.
Figure 6: The gypsy insulator increases expression of an endogenous salivary gland enhancer in the HSP70 promoter.

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Acknowledgements

We are indebted to S. Cherry for calling our attention to the phiC31 integrase system and providing critical feedback throughout the project. We also thank J. Zallen, B. Mathey-Prevot, M. Gelbart-Carey, E. Larschan, M. Levine, L. Quilter and K. Venken for helpful comments on the manuscript, and J. Philips, M. Gibson, R. Binari, J. Bateman, M. Kuroda, A. Gortchakov, A. Alekseyenko, W. Bender, M. Wolfner, S. Elledge, A. McMahon, S. Dymecki and N. Hunter for stimulating discussions. We are grateful to M. Calos, R. Nusse and M. Fish (Stanford University) for attP fly stocks, B. Fisher for attP mapping data, J. Bai (Harvard Medical School) for pUAST-luciferase, R. Lehmann (New York University Medical Center), J. Posakony (University of California San Diego), N. Dostatni (Institut Curie), G. Struhl (Columbia University), D. Arnosti (Michigan State University) and the Bloomington Stock Center for fly stocks, and FlyBase for the BLAST server. N.P. is an investigator of the Howard Hughes Medical Institute; M.M. is a fellow of the Jane Coffin Childs Memorial Fund; C.P. is a European Molecular Biology Organization (EMBO) fellow and S.E.C. receives support from the US Department of Energy contract DE-AC0376SF0098.

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M.M. and N.P. conceived the project; M.M. and C.P. conducted and interpreted the experiments; C.V. conducted embryonic injections; S.E.C. contributed the attP mapping data; M.M. wrote the manuscript; and N.P. supervised the project.

Corresponding author

Correspondence to Michele Markstein.

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Markstein, M., Pitsouli, C., Villalta, C. et al. Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes. Nat Genet 40, 476–483 (2008). https://doi.org/10.1038/ng.101

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