Ecdysone-controlled expression of transgenes

Inducible expression systems show great potential for use in human gene therapy and systems based on insect ecdysone receptors are particularly promising candidates. This article describes such systems and reviews actual and potential uses of ecdysone-controlled transgenes in vitro and in vivo. The ligand specificity of ecdysone receptor-based systems is considered, along with the safety and efficacy of the ecdysteroid and non-steroidal compounds used to activate them.     

Adenovirus-Mediated Inducible Gene Expression in Vivo by a Hybrid Ecdysone Receptor

Precise control of transgene expression would markedly facilitate certain applications of gene therapy. To regulate expression of a transferred gene in response to an exogenous compound in vivo, we modified the ecdysone-responsive system. We combined the advantages of the Drosophila (DmEcR) and the Bombyx ecdysone receptor (BmEcR) by creating a chimeric Drosophila/Bombyx ecdysone receptor (DB–EcR) that preserved the ability to bind to the modified ecdysone promoter without exogenous retinoid X receptor (RXR). In cultured cells, DB–EcR effectively mediates ligand-dependent transactivation of a reporter gene at lower concentrations of the chemical ecdysone agonist GS-E than VgRXR (DmEcR + RXR). Transgene delivery in vivo was achieved by intramyocardial injection of recombinant adenovirus vectors in adult rats. Upon stimulation with GS-E, DB–EcR potently (>40-fold induction) activated gene expression in vivo while VgRXR was not induced. This hybrid ecdysone receptor represents an important new tool for in vivo transgene regulation with potentially diverse applications in somatic and germline transfer.     

Molecular dissection of cardiac repolarization by in vivo Kv4.3 gene transfer

Heart failure leads to marked suppression of the Ca(2+)-independent transient outward current (I(to1)), but it is not clear whether I(to1) downregulation suffices to explain the concomitant action potential prolongation. To investigate the role of I(to1) in cardiac repolarization while circumventing culture-related action potential alterations, we injected adenovirus vectors in vivo to overexpress or to suppress I(to1) in guinea pigs and rats, respectively. Myocytes were isolated 72 hours after intramyocardial injection and stimulation of the ecdysone-inducible vectors with intraperitoneal injection of an ecdysone analog. Kv4.3-infected guinea pig myocytes exhibited robust transient outward currents. Increasing density of I(to1) progressively depressed the plateau potential in Kv4. 3-infected guinea pig myocytes and abbreviated action potential duration (APD). In vivo infection with a dominant-negative Kv4. 3-W362F construct suppressed peak I(to1) in rat ventriculocytes, elevated the plateau height, significantly prolonged the APD, and resulted in a prolongation by about 30% of the QT interval in surface electrocardiogram recordings. These results indicate that I(to1) plays a crucial role in setting the plateau potential and overall APD, supporting a causative role for suppression of this current in the electrophysiological alterations of heart failure. The electrocardiographic findings indicate that somatic gene transfer can be used to create gene-specific animal models of the long QT syndrome.     

Developmental profiles and ecdysone regulation of the mRNAs for two ecdysone receptor isoforms in the Mediterranean fruit fly Ceratitis capitata

Using 5' RACE with specific primers for the ecdysone receptor B1 isoform of the Mediterranean fruit fly (medfly), Ceratitis capitata, we isolated a cDNA clone encoding the specific region of the medfly ecdysone receptor A isoform (CcEcR-A). The CcEcR-A-specific region was very similar to the EcR-A-specific region of Drosophila melanogaster and less similar to the EcR-A-specific regions of Lepidoptera. The developmental expression of both CcEcR-A and CcEcR-B1 mRNAs was studied in whole animals, salivary glands and ovaries by RT-PCR, using isoform-specific primers. Both CcEcR mRNAs are present in very early embryos, decrease to very low levels during the first hours of embryogenesis and are highly expressed in all consequent embryonic stages. During metamorphosis both isoforms are present showing two peaks; the first at the larval-prepupal transition and the second during the second half of prepupal development. These peaks are correlated with the two puffing cycles and the two major 20-hydroxyecdysone (20E) increases that occur during medfly metamorphosis. CcEcR-B1 mRNA was directly induced in larval salivary glands in vitro by 20E, even at very low concentrations of the hormone, while CcEcR-A mRNA was slightly induced only by high 20E concentrations and in the absence of a protein synthesis inhibitor. During oogenesis, the CcEcR mRNAs were expressed synchronously, peaking at the beginning of both previtellogenic and vitellogenic phases.     

Analysis of the Drosophila melanogaster anti‐ovarian response to honey bee queen mandibular pheromone

Queen mandibular pheromone (QMP) is a potent reproductive signal to which honey bee workers respond by suppressing their ovaries and adopting alloparental roles within the colony. This anti‐ovarian effect of QMP on workers can, surprisingly, be induced in other insects, including fruit flies, in which females exposed to synthetic QMP develop smaller ovaries with fewer eggs. In this study, we use the Drosophila melanogaster model to identify the components of synthetic QMP required for the anti‐ovarian effect. We found that virgin females respond strongly to 9‐oxo‐2‐decenoic acid and 10‐hydroxy‐2‐decenoic acid (10HDA), suggesting that the decenoic acid components of QMP are essential for the anti‐ovarian response. Further, a nuclear factor of activated T‐cells reporter system revealed neurones expressing the olfactory receptors Or‐56a, Or‐49b and Or‐98a are activated by QMP in the antenna. In addition, we used olfactory receptor GAL4 drivers and a neuronal activator (a neuronal activating bacterial sodium channel) to test whether the candidate neurones are potential labelled lines for a decenoic acid response. We identified Or‐49b as a potential candidate receiver of the 10HDA signal. Finally, the anti‐ovarian response to synthetic QMP is not mediated by decreasing the titre of the reproductive hormones ecdysone and juvenile hormone.     

MicroRNA miR‐252 targets mbt to control the developmental growth of Drosophila

Developmental growth is an intricate process involving the coordinated regulation of the expression of various genes, and microRNAs (miRNAs) play crucial roles in diverse processes throughout animal development. The ecdysone‐responsive miRNA, miR‐252, is normally upregulated during the pupal and adult stages of Drosophila development. Here, we found that overexpression of miR‐252 in the larval fat body decreased total tissue mass through a reduction in both cell size and cell number, causing a concomitant decrease in larval size. Furthermore, miR‐252 overexpression led to a delayed larval‐to‐pupal transition with defective anterior spiracle eversion, as well as a decrease in adult size and mass. Conversely, adult flies lacking miR‐252 showed an increase in mass compared with control flies. We found that miR‐252 directly targeted mbt, encoding a p21‐activated kinase, to repress its expression. Notably, co‐overexpression of mbt rescued the developmental and growth defects associated with miR‐252 overexpression, indicating that mbt is a biologically relevant target of miR‐252. Overall, our data support a role for the ecdysone/miR‐252/mbt regulatory axis in growth control during Drosophila development.