The limited availability of Cre driver lines in the mouse for manipulating the expression of genes in the dimensions of both time and space has severely hampered biomedical research progress. These lines are required for investigators to be able to perform gene overexpression, gene misexpression, and genesilencing experiments in the normal mouse as well as gene-rescue experiments in mutant organisms. Although defined regulatory elements of some genes could, in principle, be used to drive Cre expression with control over the spatial dimension, the regulatory elements that are used to express native genes are numerous, complex, and difficult to dissect into individual elements. In addition, there are very few genes that are expressed in the mouse brain in a very limited spatial domain, such that classical knock in approaches are not anticipated to provide precise spatial specificity on expression. An alternative strategy developed by investigators working with other model organisms, particularly Drosophila, is enhancer trapping. In this strategy, a DMA segment carrying a minimal promoter that can promote the expression of a gene of interest is integrated into the genome at random in transgenic animals, so that any random integrant usurps the activity of a nearby enhancer and this causes the gene of interest to be expressed in time and space according to the determinants of the nearby enhancer. Such strategies have populated the Drosophila research field with thousands of enhancer trap lines that express Gal4 or inducible Gal4 derivatives in virtually any tissue and cell type of the organism and these reagents are the basis for uncountable discoveries. We propose here, a similar strategy in the mouse. We will construct and validate over the next 5 years more than 2500 new enhancer trap lines in the mouse that carry inducible forms of Cre. We anticipate that between 1 and 5 percent of these lines will show specific Cre expression in the brain in both time and space. The project will be performed at the Baylor College of Medicine, known for its emphasis on and excellence in mouse genetics. The project will include investigators that have developed new and inducible enhancer trap vectors in Drosophila and used these to perform large screens for brain expression patterns, outstanding molecular biologists to construct new enhancer trap vectors carrying inducible versions of Cre, mouse geneticists that have led in the construction of hundreds of new mouse transgenic/knock-out models, and experts in the histological analysis of gene expression in the mouse brain using high-throughput, robotically-based workstations for RNA in situ hybridization and other types of histological procedures. The efficiency of obtaining new Cre driver lines offering experimenter control over expression and time and space will be much greater using the random enhancer trapping-based approach compared to strategies for first defining appropriate regulatory elements and/or knocking Cre into defined genes. This is the lesson learned from Drosophila genetics and there is every reason to expect it to hold in the mouse.

U01MH078843

2006-09-12

2011-08-31