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| Currently I am working as Post Doctoral Scholar in Dr. S. R. Palli's lab, Department of Entomology, University of Kentucky, Lexington, KY, USA. I have joined this University on January, 2005. I am working mainly for the "Development and characterization of Ecdysone based gene switches in mammalian system for medical use". |
| Ecdysteroids are signaling molecules wide spread in the animals as well as in the plant kingdom. However, they do not occure naturally in vertebrates, a feature that makes them suitable as ligands in medical gene switch applications due to the reduced likelihood of pleitropic effects. Insect EcR can hetero-dimerize with retinoid X receptor (RXR) and transactivate genes that are placed under the control of Ecdysone responce element (EcRE)in various cellular backgrounds including mammalian cells. A chimeric protein composed of GAL4 DNA binding protein fused to an Ecdysone receptor protein (Ligand binding domain) and a VP16 activation domain fused with retinoid X receptor (RXR) along with the gene of interest under the control of a responce element here EcRE) not recognised by a natural nuclear receptors, are main components of EcR gene switches. Binding of specific inducer to the receptor dimer leads to its activation and consequently to the transcriptional up-regulation of any gene interest located downstream of the synthetic responce element. An Optimal gene switch should have low or no basal expression in the absence of ligand, high induced expression in the presence of ligand, rapid switch off responce after the removal of the inducer and specific responce to the inducer with no pleiotropic effects. The current version of EcR based gene switches does not have all the desirable characteristics of an optimal gene switch like high background expressions in the absence of ligand, low sensitivity with the ligand, and pleiotropic effects of ligands and or the gene switch compounds. To overcome these problem, the present investigation was carried out with the following objectives. 1. Improvemnt of gene switches to an optimal gene switch by mutations in the ligand binding pocket of EcR for low background expressions, high ligand sensitivity. 2. Developing an improved two-hybrid and single receptor gene switches. 3. To study the mechanism and functioning of the gene switches by Ligand binding and ChIP assays. 4. To study the possible pleiotropic effectes of various ligands using for EcR gene switches and the gene switch compounds by Micro-array and QRT-PCR. Achievements: 1. Pleiotropic effects of 20 different ligands and gene switch compunds were analysed. 2. An efficient singel-receptor gene switch was developed (VGEtvy) from mutant EcR. 3. An efficient two-hybrid switch with high sensitivity was developed. 4. The functioning of EcR gene switch was analysed. |