Doxycycline-induced expression of sense and inverted-repeat constructs modulates phosphogluconate mutase (Pgm) gene expression in adult Drosophila melanogaster

Michael J Allikian¹, Denise Deckert-Cruz², Michael R Rose², Gary N Landis¹ and John Tower¹

¹Molecular and Computational Biology Program, Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089-1340, USA

²Department of Ecology and Evolutionary Biology, University of California, Irvine, CA 92717, USA

author email corresponding author email

Genome Biology 2002, 3:research0021.1-0021.10doi:10.1186/gb-2002-3-5-research0021


Published:	15 April 2002

Subject areas: Genetics, Methods, Development

Abstract

Background

A tetracycline-regulated (conditional) system for RNA interference (RNAi) would have many practical applications. Such a strategy was developed using RNAi of the gene for phosphogluconate mutase (Pgm). Pgm is a candidate lifespan regulator: Pgm^S allele frequency is increased by selection for increased lifespan, whereas Pgm^M and Pgm^F allele frequencies are decreased.

Results

The Pgm alleles were cloned and sequenced and were found to differ by amino-acid substitutions consistent with the relative electrophoretic mobilities of the proteins. The 'tet-on' doxycycline-regulated promoter system was used to overexpress Pgm^S in a wild-type (Pgm^M) background. Enzyme activity increases of two- to five-fold were observed in five independent transgenic lines. Tet-on was also used to drive expression of an inverted-repeat fragment of Pgm coding region. The inverted-repeat transcript was expected to form a dsRNA hairpin, induce RNAi, and thereby reduce endogenous Pgm gene expression at the RNA level. Endogenous Pgm RNA levels in adult flies were found to be reduced or eliminated by doxycycline treatment in five independent inverted-repeat transgenic lines. Our results show that doxycycline-regulated expression of inverted-repeat constructs can cause a conditional reduction in specific gene expression. The effect of sense and inverted-repeat construct expression on lifespan was assayed in multiple transgenic lines. Under the conditions tested, altered Pgm gene expression had no detectable effect on adult Drosophila lifespan.

Conclusions

A system for conditional RNAi in Drosophila adults shows promise for assay of gene functions during aging. Our results indicate that Pgm does not have a simple strong effect on longevity.