Apoptosis induced by Pseudomonas exotoxin: A sensitive and rapid marker for gene delivery in vivo

The rapid progress in gene therapy has expanded our ability to alter geneti c structure, necessitating the development of methods for detecting the act ivity of new vectors, The central concept of a reporter gene is simple: it is a defined nucleotide sequence, which when introduced into a biological s ystem, yields a readily measurable phenotype on expression, This provides a convenient parameter that is correlated to the molecular events associated with genetic expression. In this study we demonstrate that Pseudomonas exo toxin A (PE) can serve as a sensitive reporter gene to detect gene expressi on in single cells of mouse lung on cationic liposome delivery of RE-encodi ng DNA in vivo. Furthermore, we show that PE expression can be detected as early as 2 hr after systemic gene delivery in lungs of recipient mice. We c ompared PE with the widely used P-galactosidase gene for this purpose. PE i nduces apoptosis that can be detected by TdT end labeling of DNA fragments (TUNEL assay) Since few expressed PE molecules are necessary to trigger the apoptosis cascade, the minimal amount of RE-encoding plasmid DNA needed fo r detection of apoptotic cells after systemic delivery was 0.1 mu g per ani mal compared with at least 1 mu g for the beta-galactosidase-encoding plasm id DNA, The maximum number of apoptotic cells detected in lungs was about 1 5-20 times higher than the maximum number of P-galactosidase-positive cells . Specificity of apoptosis due to PE expression on delivery of the PE-encod ing plasmid was shown by prevention of the apoptotic cascade by the caspase inhibitor Z-VAD-fmk.