The goal of this Phase II proposal is to develop systems for transient, stable, and transgenic expression of nearly any DNA sequence, particularly those that are impossible to clone into current vectors. Primary targets include genes containing long trinucleotide repeats, toxic ORFs, and very AT-rich regions of up to 50 kb. The systems will be based on a novel linear cloning vector. Variants will be developed for conventional transfection of eukaryotic cells and for generation of transgenic cell lines. In addition, we will incorporate the linear vector into a simple and robust method of transient expression, based on a modified Vesicular Stomatitis Virus ("VSV-T7"). Together these methods will allow mammalian expression and analysis of nearly any DNA sequence, in vivo or in vitro, including those that contain highly repetitive regions, have a high AT or GC content, or encode proteins that are toxic or deleterious to bacterial or mammalian hosts. This system will significantly expand the repertoire of genes that can be expressed and studied in mammalian cells, including cDNAs that have eluded cloning in high-throughput projects. Expression of otherwise toxic or unstable DNAs may lead to important advances in basic research and medical science. The systems described here will facilitate research on human Repeat Expansion Diseases, such as Fragile X Syndrome, Muscular Dystrophy, Friedreich ataxia, Huntington's Disease, Spinocerebellar ataxia, etc. They will allow cloning and expression of large eukaryotic genes, e.g., to analyze alternative splicing and co-express multiple proteins. They will also allow expression of proteins from pathogenic organisms, which often have very AT-rich genomes, such as Plasmodium sp., Pneumocystis carinii, and others. Importantly, the VSV-T7 system will simplify transient expression and make it amenable to high-throughput screening. PUBLIC HEALTH RELEVANCE: The proposed SBIR application, "Novel systems for mammalian expression, integration, and knockout of previously 'unclonable'genes", aims to provide a simple and efficient method to express high levels of recombinant proteins in mammalian cell cultures. This work will facilitate expression of numerous proteins that are currently refractory to cloning, and its ease of use and robust expression will enable high-throughput production of proteins in mammalian cells.