Abstract
Background
Phage ϕC31 integrase has emerged as a potent tool for achieving long‐term gene expression in different tissues. The present study investigated the activity of ϕC31 integrase in murine lungs.
Methods
Transfections in murine alveolar epithelial (MLE12) cells were performed with Lipofectamine 2000. For in vivo gene delivery, DNA was complexed with polyethylenimine (PEI) and PEI‐DNA complexes were injected intravenously into mice. Expression of luciferase in mice was monitored by in vivo bioluminsecence imaging. Genomic integration and integration into a previously described ‘hotspot’ were confirmed by polymerase chain reaction (PCR).
Results
ϕC31 integrase mediated intramolecular recombination between wild‐type attB and attP sites in MLE12 cells. Long‐term gene expression could be observed in MLE12 cells in the presence of integrase without any selection pressure. Long‐term expression of luciferase after intravenous injection of PEI‐DNA complexes could be observed only in the lungs of mice which were co‐injected with the integrase‐encoding plasmid. Increased amounts of integrase plasmid and administration of a second dose had no effect on the level of luciferase expression achieved with a single dose, which was three orders of magnitude lower than the values observed on ‘day 1’ post application. Genomic integration of the transgene in the mouse lungs was confirmed by PCR. Seven out of the fifteen treated mice showed integration at the mpsL1 site, a previously described ‘hot spot’ from liver.
Conclusions
These results provide evidence for the activity of ϕC31 integrase in lungs but also emphasize the need for optimization of the system to maintain long‐term gene expression at high levels. Copyright © 2007 John Wiley & Sons, Ltd.