The goal of the work proposed here is to complete development of a highly versatile genetrap system, to begin trapping genes with this system, and to demonstrate its utility in characterizing trapped genes. Fundamentally, our genetrap system is comprised of three tiers of constructs, referred to as controller, effector and responder. The controller is designed to trap genes in ES cells. The effector and responder, each with several specialized versions, will be used to make stock transgenic mice. To perform functional studies of a trapped gene, mice bearing the trapped gene will be crossed to mice containing desired versions of the effector and responder transgenes. In the progeny that possess all three tiers of constructs, interaction among the three tiers can be conditionally triggered with drug treatment to facilitate the intended functional analysis. Such a 3-tiered system can facilitate a diverse array of studies, mainly because different combinations of the three tiers can accommodate disparate tasks. Major capabilities of the system include gene expression monitoring, fate mapping, conditional cell ablation, and conditional knockout, among others.
Our specific aims are: 1) To construct key DNA vectors of the 3-tiered system, and to systematically test them in cell culture. 2) To generate a total of 2500 genetrap clones with the system in five different embryonic stem cell lines (including three CS7BJJ6 lines), and to obtain sequence tags of the insertion sites. 3) To produce two basic sets of transgenic mice: a standard set of stock mice transgenic for particular versions of the effector and responder, and a set of genetrap mice made from 15 selected genetrap clones. 4) To test the utility of the 3-tiered system in mice, focusing on the ability of the system to perform the following tasks: gene disruption, expression monitoring, fate mapping, and conditional cell ablation. Collectively, these experiments will pave the way for our ultimate goal, which is to use the 3-tiered system to create a widely applicable set of functional genomics resources for the mouse community. These will include a large genetrap library, and a standard collection of transgenic mice useful for systematic interrogation of diverse trapped genes.
