The objectives of this proposal is to develop a novel eukaryotic expression system that can provide significantly higher gene expression than the current vectors in a broad range of cell types. The system is based on the fact that hnRNP L can stimulate polyadenylation when binding to its optimal binding sites. Specifically, to achieve the objectives, the molecular mechanism of hnRNP L facilitated expression enhancement (LFEE) will be elucidated by in vitro assays, which will also work as easy tests for effects of different hnRNP L binding sites and their combinations. The in vitro results will be correlated with in vivo expression assays by including LFEE elements in reporter constructs. The effect of co-expression of hnRNP L as a built-in feature will eventually be analyzed in vivo as well. Once these specific aims are accomplished, novel expression vectors will be assembled as LFEE or LFEE2 (with hnRNP L co-expression) that contain combinations of optimal hnRNP L binding sites at chosen places that gave the highest expression level, and compatible promoter, polylinker, and poly(A) signal, etc. The commercialization of such vectors should provide a very useful tool for biopharmaceutical production, transgenics, DNA vaccine and gene therapy.
This proposal intends to develop a novel expression system for application in a broad range of eukaryotic cells. This technology is aimed at lowering the cost of recombinant protein therapeutics. Because of the unique mechanism underlying this technology, its success may also have a great impact in the fields of transgenics, gene therapy, and DNA vaccine.
