phiC31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study aimed at optimizing elements of phiC31 integrase system for alveolar type II cells. Luciferase and beta-galactosidase activities were measured at different time points post transfection. 5-Aza-2'deoxycytidine (AZA) and trichostatin A (TSA) were used to inhibit DNA methyltransferase and histone deacetylase complex (HDAC) respectively. In A549 cells, expression of the integrase using a CMV promoter resulted in highest integrase activity, whereas in MLE12 cells, both CAG and CMV promoter were equally effective. Effect of polyA site was observed only in A549 cells, where replacement of SV40 polyA by bovine growth hormone (BGH) polyA site resulted in an enhancement of integrase activity. Addition of a C-terminal SV40 nuclear localization signal (NLS) did not result in any significant increase in integrase activity. Long-term expression studies with AZA and TSA, provided evidence for post-integrative gene silencing. In MLE12 cells, both DNA methylases and HDACs played a significant role in silencing, whereas in A549 cells, it could be attributed majorly to HDAC activity. Donor plasmids comprising cellular promoters ubiquitin B (UBB), ubiquitin C (UCC) and elongation factor 1alpha (EF1alpha) in an improved backbone prevented post-integrative gene silencing. In contrast to A549 and MLE12 cells, no silencing could be observed in human bronchial epithelial cells, BEAS-2B. Donor plasmid coding for murine erythropoietin under the EF1alpha promoter when combined with phiC31 integrase resulted in higher long-term erythropoietin expression and subsequently higher hematocrit levels in mice after intravenous delivery to the lungs. These results provide evidence for cell specific post integrative gene silencing with phiC31 integrase and demonstrate the pivotal role of donor plasmid in long-term expression attained with this system.
Animals ; Bacteriophages/*genetics ; Cell Line ; Chick Embryo ; Female ; Gene Expression ; Gene Silencing ; Gene Therapy ; Genes, Reporter ; Genetic Vectors/*genetics ; Humans ; Integrases/*genetics ; Mice ; Mice, Inbred BALB C ; Plasmids/genetics ; Pneumocytes/*metabolism ; Promoter Regions, Genetic ; Streptomyces/*virology ; Transfection

Alveolar type II cells are main target of hyperoxia-induced lung injury. The authors investigated whether lysosomal protease, cathepsin B (CB), is activated in fetal alveolar type II cells in the transitional period from the canalicular to saccular stages during 65%-hyperoxia and whether CB is related to fetal alveolar type II cell (FATIIC) death secondary to hyperoxia. FATIICs were isolated from embryonic day 19 rats and exposed to 65%-oxygen for 24 h and 36 h. The cells exposed to room air were used as controls. Cell cytotoxicity was assessed by lactate dehydrogenase-release and flow cytometry, and apoptosis was analyzed by TUNEL assay and flow cytometry. CB activity was assessed by colorimetric assay, qRT-PCR and western blots. 65%-hyperoxia induced FATIIC death via necrosis and apoptosis. Interestingly, caspase-3 activities were not enhanced in FATIICs during 65%-hyperoxia, whereas CB activities were greatly increased during 65%-hyperoxia in a time-dependent manner, and similar findings were observed with qRT-PCR and western blots. In addition, the preincubation of CB inhibitor prior to 65%-hyperoxia reduced FATIIC death significantly. Our studies suggest that CB activation secondary to hyperoxia might have a relevant role in executing the cell death program in FATIICs during the acute stage of 65%-hyperoxia.
Animals ; Caspase 3 ; Cathepsin B/*metabolism ; Cell Death ; Cell Hypoxia ; Enzyme Activation ; Female ; In Situ Nick-End Labeling ; L-Lactate Dehydrogenase/analysis ; Lung/metabolism ; Necrosis/metabolism ; Oxygen ; Pneumocytes/cytology/*metabolism ; Polymerase Chain Reaction ; Pregnancy ; Pulmonary Alveoli/cytology/embryology/*enzymology ; Rats ; Rats, Sprague-Dawley