? The overall goal of the proposed research program is to develop a gene activation technology that can be applied to spatially discrete tissue subsites in the adult animal. The importance of this technology is that it should furnish a direct means to examine the biological consequences of gene expression within the context of specific tissue microenvironments. For example, the growth and progression of breast and prostate tumors are dependent upon the microenvironment in which they develop. However, it is not yet feasible to correlate the activity (or lack thereof) of a particular protein with oncogenic potential as a function of tissue microenvironment. Our strategy is based upon the ecdysteroid-driven gene expression system developed by Evans and his colleagues and further refined at the Albert Einstein College of Medicine by Pestell and his coworkers. We have prepared a light-activatable form of ecdysone that, in combination with spot illumination, can drive protein expression in any desired subset of cells present in a multicellular environment. We wilt prepare caged ecdysteroids that can be photochemically unleashed using two-photon illumination. The gene activation properties of these light-activatable agents will be evaluated both in cell-free and cell-based model systems and in the presence or absence of two-photon illumination. The latter technology furnishes deeper tissue penetration and a significantly reduced phototoxic load compared to single photon irradiation. Consequently, it is uniquely applicable to whole animal systems. We will evaluate the efficacy of light-induced protein expression in two distinct, but commonly employed, animal models. The first model consists of ectopically expressed tumors in Fischer 344 rats routinely employed in the Condeelis group. The second model is a previously described triply transgenic mouse line developed in the Pestell group. ? ?
| Singer, Robert H; Lawrence, David S; Ovryn, Ben et al. (2005) Imaging of gene expression in living cells and tissues. J Biomed Opt 10:051406 |
