Developing tissue-specific inducible expression systems

grantor: University of TorontoInducible expression systems allow the external regulation of gene expression and enable gene function analysis to be performed in a dose-dependent manner. We developed an epithelium-specific tetracycline inducible system using the regulatory elements of the human cytokeratin 19 gene. High levels of expression were achieved in a dose-dependent manner in cultured lung epithelial cells, ' in vitro'. Only very low levels of expression were observed in cultured fibroblast cells. In transgenic mice we demonstrated the ability of the system to regulate inducible expression of a reporter gene in the epithelia of the upper airways and the submucosal glands. RNA analysis revealed a cryptic splicing reaction in the RNA expression of the transactivator protein. Modifications to the expression construct were made and mutants were screened for increased expression. One construct, with a modified 3' untranslated region, was capable of inducing 60% greater expression than our first generation construct. This modified construct was used to test a complimentary tetracycline regulatable inducible system. Using this system, it will be possible to target the expression of a gene of interest to the lung epithelia and controlled temporally, without pleitropic effects, by the administration of doxycycline.M.Sc