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.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Animals
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Animals, Genetically Modified
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Attachment Sites, Microbiological / genetics*
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Chromatin / physiology*
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Cytoskeletal Proteins / genetics
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Cytoskeletal Proteins / metabolism
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Drosophila Proteins / genetics
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Drosophila Proteins / metabolism
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Drosophila melanogaster / genetics*
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Drosophila melanogaster / growth & development
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Drosophila melanogaster / metabolism
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Female
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Gene Expression Regulation
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Genome, Insect / genetics
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HSP70 Heat-Shock Proteins / genetics
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HSP70 Heat-Shock Proteins / metabolism
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Insulator Elements / genetics*
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Larva / metabolism
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Molecular Sequence Data
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Myogenic Regulatory Factors / genetics
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Myogenic Regulatory Factors / metabolism
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Phenotype
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Plasmids
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Receptors, Notch / genetics
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Receptors, Notch / metabolism
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Recombination, Genetic*
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Retroviridae / genetics*
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism
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Tissue Distribution
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Transgenes / physiology*
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Wings, Animal / physiology
Substances
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ARP1 protein, S cerevisiae
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AttB protein, Drosophila
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Chromatin
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Cytoskeletal Proteins
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Drosophila Proteins
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HSP70 Heat-Shock Proteins
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Mef2 protein, Drosophila
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Myogenic Regulatory Factors
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N protein, Drosophila
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Receptors, Notch
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Saccharomyces cerevisiae Proteins
Associated data
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GENBANK/EU420016
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GENBANK/EU420017
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GENBANK/EU420018
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GENBANK/EU420019
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GENBANK/EU420020