Date of Completion
Guillermo R. Risatti; Daniel J. Gage
Field of Study
Master of Science
Two vaccinia virus (VACV) expression systems that contain elements from the lactose (lac) and the tetracycline (tet) operons of E. coli were developed to repress the expression of a reporter gene, enhanced green fluorescent protein (EGFP), in the presence of tet operon inducers. In the first system, lac and tet operon elements were arranged in a gene circuit, and in the presence of increasing concentrations of a lac operon inducer (isopropyl-β-D-thiogalactoside, IPTG), EGFP expression increased in a dose dependent manner and at high IPTG concentrations, expression reached the same levels as a positive control virus. Importantly, in the presence of increasing concentrations of the tet operon inducer (doxycycline, DOX), EGFP expression decreased in a dose dependent manner and at high concentrations of DOX, expression was repressed to the degree observed in a negative control virus that does not express EGFP. In the second system, recombinant VACVs constitutively expressing six mutant versions of the tet repressor gene (tetR) shown to bind tet operators in the presence, but not absence of inducers (reverse tetR genes), were developed. In the presence of tetracyclines (TCs), the recombinant VACVs exhibited various degrees of repression of EGFP expression, with increasing concentrations of TCs leading to EGFP repression in a dose dependent manner, and in some instances, down to the degree observed in a negative control virus. In light of the renewed interest for the use of VACV as vaccine and therapeutic cancer vectors, the repressible VACV expression system developed here can be used to tightly regulate genes essential for VACV replication, thus functioning as a built-in safety mechanism to conditionally control viral replication.
Titong, Allison, "Development of Repressible Systems to Control Gene Expression in Vaccinia Virus" (2012). Master's Theses. 334.
Paulo H. Verardi