Within the community of scientists who study the biology and application of stem cells there is a need for the controlled expression of specific gene sequences. Our overall objective is to develop recombinant gene transfer systems with two features: tight control of transgene expression and inducible transgene expression. Specifically, we will develop vectors that expand populations of multipotent hematopoietic stem cells (HSCs) both ex vivo and in vivo while maintaining their undifferentiated state. Investigators have tested a variety of strategies to accomplish this aim, but success has been hampered by the complexity of reagents required to control transgene expression. We have generated a fusion protein that combines a genetically engineered ligand binding domain specific for a synthetic """"""""near drug"""""""" and a DMA binding domain specific for a DNA sequence not found in the mammalian genome (i.e. the Gal4 response element). By cloning cDNA sequences downstream of the response element we have achieved remarkable control over transcription of several cDNA sequences. Our strategy has many advantages over currently available systems, including exquisitely tight control over transgene expression and high selectivity of our engineered ligand binding domain. Using this system, we have designed first generation retroviral constructs that will allow controlled transgene expression in genetically engineered cells. The goals of this proposal are to determine the extent that control of proviral sequences can be genetically engineered into retroviral constructs and to test the effectiveness of our inducible systems to regulate the controlled expression of cDNA sequences that regulate the growth of multipotent HSCs.
Our first aim i s to develop and assess the effectiveness of orthogonal promoters that express reporter genes (luciferase and GFP) as well as HoxB4, which has clearly been shown to be important in the expansion of HSCs.
Our second aim i s to test regulated expression of HoxB4 in HSCs transduced with our inducible vectors to enforce HSC expansion in vivo. ? ?
| Taylor, Jennifer L; Rohatgi, Priyanka; Spencer, H Trent et al. (2010) Characterization of a molecular switch system that regulates gene expression in mammalian cells through a small molecule. BMC Biotechnol 10:15 |
