Abstract
Transgenic Drosophila have contributed extensively to our understanding of nervous system development, physiology and behavior in addition to being valuable models of human neurological disease. Here, we have generated a novel series of modular transgenic vectors designed to optimize and accelerate the production and analysis of transgenes in Drosophila. We constructed a novel vector backbone, pBID, that allows both phiC31 targeted transgene integration and incorporates insulator sequences to ensure specific and uniform transgene expression. Upon this framework, we have built a series of constructs that are either backwards compatible with existing restriction enzyme based vectors or utilize Gateway recombination technology for high-throughput cloning. These vectors allow for endogenous promoter or Gal4 targeted expression of transgenic proteins with or without fluorescent protein or epitope tags. In addition, we have generated constructs that facilitate transgenic splice isoform specific RNA inhibition of gene expression. We demonstrate the utility of these constructs to analyze proteins involved in nervous system development, physiology and neurodegenerative disease. We expect that these reagents will facilitate the proficiency and sophistication of Drosophila genetic analysis in both the nervous system and other tissues.
Highlights
The ability to express an exogenous gene in a transgenic animal with control over the timing, level and pattern of expression is essential for many types of experimental analysis
The sophistication of Drosophila genetics combined with the advent of transformation with transposable elements vectors [1] followed by subsequent innovations such as the Gal4/Upstream Activation Sequence (UAS) system [2] have made Drosophila a powerful model system in which to address a multitude of biological problems including the development and function of the nervous system [3]
Drosophila transgenes, including constructs introduced by QC31 transgenesis [4], can have different levels of expression depending upon the genomic location of the landing site [9] and these differences persist even at sites of relatively high expression such as commonly used attP18 on the site on the X chromosome, attP40 site on chromosome 2 and the attP2 site on chromosome 3 [9,10]
Summary
The ability to express an exogenous gene in a transgenic animal with control over the timing, level and pattern of expression is essential for many types of experimental analysis. More recently Drosophila transgenic technology has been further improved by the ability to reproducibly target transgenes to specific genomic loci using QC31 phage integrase based DNA recombination [4]. WC31 integrase catalyzes the recombination between a phage attachment site (attP) and a bacterial attachment site (attB) [5]. For Drosophila transgenesis, an attP site is introduced into the genome using conventional transposon techniques [4]. Injected plasmids containing an attB site can integrate at this ‘landing’ or ‘docking’ site when WC31 integrase is provided from either a co-injected mRNA [4] or a transgenic source [6]. The integration event is both highly efficient and unidirectional with integrated transgenes remaining stable in the presence of integrase [7]
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