To understanding the role of a gene of interest and use it for gene therapy we need a way to control its expression in mammalian cells. The usual approach is to modulate transcription/ Accumulation of mRNA commonly require hours. We will develop a system based on the inducible translational frameshifting that can shorten the lag time from minutes to minutes. The system is associated with the production of the protein antizyme, an inhibitor of polyamine production. The antizyme mRNA consists of two over-lapping reading frames, the second of which lacks an appropriately placed AUG initiation codon; protein expression depends on 1+ translational frameshifting induced by cellular polyamines. We propose to optimize the frameshift sequence and create a system of vectors in which desired genes will be transcribed, but not translated until polyamines are introduced. Development will start with frameshift regions from diverse organisms, and fusing these to reporters that provide a convenient readout of expression. The frameshift region will be optimized through a process of directed mutation and selection. Candidate regions created in this way will be used in a proof of concept to control expression of the tumor suppressor p53.
Analysis of gene expression requires a means for conditional expression of proteins. The proposed system for controlling protein synthesis by modulating translation offers advantages compared to presently available means, which modulate transcription. A series of vectors based on the proposed technology and reagents to facilitates their use will be marketed.
