Abstract
Apoptosis is an essential process for the maintenance of normal physiology. The ability to noninvasively image apoptosis in living animals would provide unique insights into its role in normal and disease processes. Herein, a recombinant reporter consisting of beta-galactosidase gene flanked by two estrogen receptor regulatory domains and intervening Asp-Glu-Val-Glu sequences was constructed to serve as a tool for in vivo assessment of apoptotic activity. The results demonstrate that when expressed in its intact form, the hybrid reporter had undetectable beta-galactosidase activity. Caspase 3 activation in response to an apoptotic stimulus resulted in cleavage of the reporter, and thereby reconstitution of beta-galactosidase activity. Enzymatic activation of the reporter during an apoptotic event enabled noninvasive measurement of beta-galactosidase activity in living cells, which correlated with traditional measures of apoptosis in a dose- and time-dependent manner. Using a near-infrared fluorescent substrate of beta-galactosidase (9H-{1,3-dichloro-9,9-dimethylacridin-2-one-7-yl} beta-D-galactopyranoside), noninvasive in vivo imaging of apoptosis was achieved in a xenograft tumor model in response to proapoptotic therapy. Finally, a transgenic mouse model was developed expressing the ER-LACZ-ER reporter within the skin. This reporter and transgenic mouse could serve as a unique tool for the study of apoptosis in living cells and animals, especially in the context of skin biology.