Controlled transgenics via 3'UTR and site specificity

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to provide a reliable, repeatable, and comparable method for generating mouse transgenics that allows for exquisite control over expression level, genome location, and strain background. The tool set ultimately produced will be a collection of 6 bacterial artificial chromosome (BAC) vectors designed to work in complimentary hprt negative mouse embryonic stem cells. Transgenic mice can be made with much greater ease and efficiency when this modular system is combined with its stringent selectable properties. Defined placement of a transgene in the genome and control over mRNA expression will greatly impact the biological relevance of mouse transgenics bringing us closer to accurately assessing gene function and the consequences of mutation. The core technology relies on the modulation of mRNA stability using a collection of five defined 3' untranslated regions (UTRs) in conjunction with site-specific integration at the drug selectable Hprt gene locus. This system provides for a defined alteration in gene expression levels of up to 100-fold. Modification of this technology for use in BACs, opens the door for its use in making transgenics in a variety of mouse strains. A series of BACs will be constructed consisting of 5 vectors where a gene of interest can be placed under the influence of one of five different 3' UTRs, and an additional test vector. All vectors will undergo stringent testing to make sure they are performing appropriately. In phase II we will expand the technology to include additional levels of gene expression control based on the use of newly defined 3'UTR regions. A wide collection of mouse ES cell lines from different mouse strains will also be made available for use with this system.