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Bioluminescent imaging of reporter gene expression in the lungs of wildtype and model mice following the administration of PEG‐stabilized DNA nanoparticles
Author(s) -
Ziady Assem G.,
Kotlarchyk Maxwell,
Bryant Laura,
McShane Matthew,
Lee Zhenghong
Publication year - 2010
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/jemt.20855
Subject(s) - transgene , wild type , gene expression , microbiology and biotechnology , luciferase , reporter gene , isoflurane , real time polymerase chain reaction , chemistry , dna , bioluminescence imaging , gene , biology , transfection , biochemistry , mutant , organic chemistry
DNA nanoparticles (DNPs) formed by compacting DNA with polyethyleneglycolylated poly‐ L ‐lysine are a nonviral vector shown to be safe and efficacious in animals and humans. To extend our capabilities of assessing the efficacy and duration of expression achieved by DNPs, we tested the utility of bioluminescent imaging (BLI) of transgene expression in wildtype and cystic fibrosis (CF) mouse models. We tested the effect of route of administration, mouse coat color, anesthesia, dose, and promoter sequence on the level and duration of expression. Furthermore, we investigated the correlation between imaging and direct analysis of luciferase expression in lung homogenates. We found that intratracheal instillation, and the use of deep and prolonged anesthesia with avertin produced significantly higher expression compared with intranasal administration, and the use of lighter anesthesia with isoflurane. Although similar expression was observed for both dark and light coat animals, imaging signal intensity was attenuated in mice with dark fur. Furthermore, good correlation between imaging and direct homogenate analysis was observed for single dose ( r = 0.96), and dose response studies in wildtype ( r = 0.82) and CF mice ( r = 0.87). Finally, we used imaging to track gene expression over a 56‐day time course. We found that the human ubiquitin B promoter gives stable transgene expression up to 49 days following nanoparticle administration, while expression with the cytomegalovirus promoter diminished after 2 days and returned to background levels by day 14. Taken together, our results demonstrate that BLI is an effective and useful modality for measuring gene expression conferred by DNPs in the lung. Microsc. Res. Tech. 73:918–928, 2010. © 2010 Wiley‐Liss, Inc.