Our preliminary results from the R01 grant that this application intends to support have revealed multiple genes that are differentially expressed in naive versus effector CD8+ T cells. The methodology of utilizing T cells transgenic for the Coxsackie and adenovirus receptor (CAR) transduced with adenoviral vectors has generated a powerful model for introducing wild type or mutant genes into resting post-thymic T cells for mechanistic studies. However, there are several attractive candidate genes the expression of which we would like to down-regulate in normal resting T cells, either in the naive state or following differentiation However, technologies to achieve this are lacking. The overall goal of the current proposal is develop and implement two strategies to eliminate expression of genes in normal post-thymic T cells at any desired differentiation state. Each approach takes advantage of the high transduction efficiency achieved with adenoviral vectors and CARTg T cells. In Specific Aim 1 we will develop and apply antisense adenoviral vectors to down-regulate expression of specific genes in CARTg T cells. The glucose transporter GLUT1 and the cytoskeletal protein calpactin/Annexin II will be studied first. In Specific Aim 2 we will develop and implement a system for conditional gene targeting using a Cre adenovirus and CARTg T cells. A LoxP/LacZ reporter mouse will be used as proof of concept, and conditional targeting constructs will be produced for GLUT1 and calpactin/Annexin II. Collectively, these technologies will expand greatly our ability to study the function of specific gene products in controlling functions of normal peripheral CD8+ T cells, with potential application toward a multitude of problems in T cell biology.