IN-SITU HISTOCHEMICAL DETECTION OF BETA-GALACTOSIDASE ACTIVITY IN LUNG - ASSESSMENT OF X-GAL REAGENT IN DISTINGUISHING LACZ GENE-EXPRESSIONAND ENDOGENOUS BETA-GALACTOSIDASE ACTIVITY

Bacterial lacZ is one of the most commonly used reporter genes for ass essing gene transfer to lung, However, lung contains endogenous beta-g alactosidase (beta-Gal), which can confound estimation of exogenous la cZ expression by histochemical techniques (i.e., X-Gal) for in situ de monstration of enzyme activity, We investigated several parameters of the X-Gal reaction, including time and temperature of X-Gal exposure a s well as lung tissue processing and fixation techniques, and found th at none of these could be used to distinguish between endogenous and e xogenous beta-Gal activities, The mammalian and bacterial beta-Gal enz ymes, however, have pH optima in the acidic and neutral ranges, respec tively, Exposing whole lung, lung minces, or mounted frozen sections o f lung to X-Gal at mildly alkaline pH (pH 8.0-8.5), minimized detectio n of endogenous activity in lungs from a variety of species while pres erving that resulting from bacterial enzyme activity in a transgenic m ouse expressing lacZ, This technique was also useful in distinguishing endogenous activity from that resulting from adenovirus-mediated lacZ gene transfer to diploid lung fibroblasts in primary culture, An appr opriate buffer that maintains the desired pH throughout the duration o f X-Gal exposure must be used.