Alteration of Drosophila life span using conditional, tissue-specific expression of transgenes triggered by doxycyline or RU486/Mifepristone

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Abstract

The conditional systems Tet-on and Geneswitch were compared and optimized for the tissue-specific expression of transgenes and manipulation of life span in adult Drosophila. Two versions of Tet-on system reverse-tetracycline-Trans-Activator (rtTA) were compared: the original rtTA, and rtTAM2-alt containing mutations designed to optimize regulation and expression. The rtTAM2-alt version gave less leaky expression of target constructs in the absence of doxycyline, however the absolute level of expression that could be achieved was less than that produced by rtTA, in contrast to a previous report. Existing UAS-rtTAM2-alt insertions were re-balanced, and combined with several tissue-general and tissue-specific GAL4 driver lines to yield tissue-specific, doxycyline-inducible transgene expression over three orders of magnitude. The Geneswitch (GS) system also had low background, but the absolute level of expression was low relative to Tet-on. Consequently, actin5C-GS multi-insert chromosomes were generated and higher-level expression was achieved without increased background. Moderate level over-expression of MnSOD has beneficial effects on life span. Here high-level over-expression of MnSOD was found to have toxic effects. In contrast, motor-neuron-specific over-expression of MnSOD had no detectable effect on life span. The results suggest that motor-neuron tissue is not the essential tissue for either MnSOD induced longevity or toxicity in adult males.

Introduction

Conditional gene expression systems are a powerful tool for the study of Drosophila aging mechanisms (Aigaki et al., 2002, Giannakou et al., 2004, Hwangbo et al., 2004, Tower, 2000). The primary advantages of these systems are that experimental and control groups are genetically identical, produced from the same cross, and share developmental conditions, and that the experimenter can decide when in the life cycle to turn transgene expression on and off. The basic principle of all conditional systems is that an environmental stimulus is used to activate expression of a gene of interest. Here two conditional systems were compared and optimized for the tissue-specific expression of transgenes in Drosophila. The “Tet-on” system uses the tetracycline derivative doxycyline (DOX) as the stimulus, and the “Geneswitch” system uses the drug RU486/Mifepristone.

The reverse-tetracycline transactivator (rtTA) is an artificial transcription factor that was developed to allow doxycyline-regulated transgene expression in eukaryotes (Gossen and Bujard, 1995, Kistner et al., 1996). The rtTA protein consists of the DNA binding domain of the Escherichia coli reverse-tet-repressor, fused to the transcriptional activation domain of the herpes simplex virus VP16 transcription factor. The result is an artificial transcription factor that will bind to its target site, the tet-operator sequence (TetO) and activate transcription of an adjacent promoter only in the presence of DOX or other tetracycline derivatives (the Tet-on system). The Tet-on system was introduced into Drosophila melanogaster by driving expression of rtTA with the tissue-general actin5C promoter (Fig. 1a). A synthetic target promoter (the Tet-on promoter) was produced by placing 7 TetO sites adjacent to a truncated or “core” promoter derived from the hsp70 or other genes. This system yielded up to a 100-fold induction of reporter proteins such as β-galactosidase in adult male flies without a detectable decrease in life span (Bieschke et al., 1998).

The rtTAM2-alt form of the rtTA transactivator was created with multiple mutations designed to yield greater expression and tighter regulation by DOX. The rtTAM2-alt transactivator was also introduced into Drosophila under the control of the actin5c promoter (Stebbins et al., 2001). While it was reported that rtTAM2-alt yielded higher levels of transgene expression than the original rtTA, a meaningful comparison was not possible, due to the lack of detectable rtTA activity in that study. In addition, the researchers developed a tissue-specific Tet-on system by combining rtTAM2-alt with GAL4/UAS. In the GAL4/UAS system, a tissue specific promoter or enhancer drives expression of the yeast transcription factor GAL4 in one transgenic construct. GAL4 then binds to an upstream activating sequence (UAS) site in the promoter of a second target construct and activates transcription (Brand and Perrimon, 1993, Seroude, 2002, Seroude et al., 2002). Stebbins and coworkers combined these two systems by creating a bridge construct where UAS promoter drives expression of rtTAM2-alt (Stebbins et al., 2001). In the combined system the “GAL4 driver” chromosome yields GAL4 expression only in specific tissues (Fig. 1b). The GAL4 binds to the UAS promoter in the bridge construct, which drives rtTAM2-alt expression. The rtTAM2-alt will in turn bind to the TetO sites in a third target construct, but only in the presence of DOX. The result is transgene expression that is both tissue-specific and conditional.

A mobile Tet-on promoter has been developed in order to increase the number of genes that can be regulated by the Tet-on system. A Drosophila P type transposable element was constructed, called PdL, with a DOX-inducible promoter directed outward from its 3′ end (Landis et al., 2001). Transposable elements with outwardly directed promoters, like PdL, have been shown to be powerful tools for creating mutations by over-expression and/or mis-expression of genes near their insertion site (Rorth et al., 1998). When PdL transposes into a fly chromosome, it can cause DOX-dependent over-expression of a gene up to thousands of base-pairs downstream of the insertion site. It is estimated that up to a third of PdL insertions cause the over-expression of a downstream gene. Gene over-expression caused by PdL frequently, but not always, results in a conditional mutant phenotype.

The Geneswitch system also allows for conditional gene expression in Drosophila (Osterwalder et al., 2001, Roman and Davis, 2002, Roman et al., 2001). In this case the transactivator protein, Geneswitch, is a fusion of the DNA binding and transcriptional activation domains of yeast GAL4 with the regulatory domain of the human progesterone receptor. Geneswitch will bind to a UAS site and activate transcription only in the presence of progesterone, or analogs such as RU486. In this study the Tet-on and Geneswitch systems have been compared and optimized for use in Drosophila aging research.

Section snippets

Materials and methods

Drosophila melanogaster strains are as previously described (Table 1) (Bieschke et al., 1998, Landis et al., 2001, Lindsley and Zimm, 1992). Northern blot procedures were as previously described (Landis et al., 2001). Briefly, 30 adult males of the indicated genotypes were cultured in the presence of drug, and 30 in the absence of drug, for one week with transfer to fresh food vials every-other day. Total RNA was extracted from the flies using RNAqueous kit (Ambion), fractionated on 1% agarose

Comparison of the Tet-on actin5C-rtTA driver lines

The actin5C-rtTA constructs (rtTA) yield a tissue-general pattern of expression in the adult fly (Bieschke et al., 1998). However, it is important to characterize multiple transgenic lines, since the insertion site can affect both the absolute level and the tissue specificity of expression. Several rtTA lines have previously been compared using β-galactosidase assays to test their ability to activate a lacZ reporter construct in adult males, and a range of activities was observed (Bieschke et

Discussion

Both the Tet-on system and Geneswitch systems were found to have specific advantages for manipulating adult life span. The Tet-on system has a generally higher level of expression than the Geneswitch system, and a greater potential range of tissue specificity via combination with existing GAL4/UAS lines. A potential advantage of the Geneswitch system is that the drug RU486 has no significant effect on life span. DOX can sometimes have a small positive effect (∼0–4%) depending on the particular

Acknowledgements

We thank Cheryl Teoh and Jie Shen for assisting with the lacZ expression assays. This work was supported by a grant from the Department of Health and Human Services to J.T. (AG11833). A.B. was supported in part by NIA training grant (AG00093).

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    Present address: Amylin Pharmaceuticals, 9360 Towne Centre Drive, Ste 110, San Diego, CA 92129, USA.

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