We have developed a hybrid vector that combines the high transduction efficiency of a gene-deleted adenoviral vector and the integration machinery of the bacteriophage-derived integrase PhiC31 for stable transduction and limited integration sites. We based our system on a two-vector system in which the transgene expression cassette is circularized from a helper-dependent vector by Flp-mediated recombination, followed by PhiC31-mediated integration. Integration of the transgene expression cassette from the adenoviral vector resulted in 5-fold higher transgene expression levels in the active PhiC31 group compared to the control group, which received a mutated and inactive version of PhiC31. We confirmed transgene integration into the previously described mpsL1 hot spot of integration by polymerase chain reaction analyses of DNA isolated from mouse livers. In addition, we cloned 40 integration sites. The hot spot mpsL1 was detected only once, and 44% of all integration events were found to be present in gene regions. With further optimization, this system represents a new tool for gene therapy protocols that may offer an alternative to gene therapy approaches based on random integrating viral vectors.

BACKGROUND

Phage phiC31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study investigated the activity of phiC31 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

phiC31 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 phiC31 integrase in lungs but also emphasize the need for optimization of the system to maintain long-term gene expression at high levels.

BACKGROUND

Achieving long-term gene expression in kidney will be beneficial for gene therapy of renal and congenital diseases, genetic studies constructing animal disease models, and the functional analysis of disease-related genes.

OBJECTIVE

The purpose of this study was to develop an in vivo long-term gene expression system in murine kidney using φC31 integrase.

METHODS

Gene expression in cultured RENCA, TCMK-1, and HEK293 cells was assessed. The long-term in vivo gene expression system in the kidney was achieved by co-transfecting 5 µg of pORF-luc/attB as a donor plasmid and 20 µg of pCMV-luc as a helper plasmid into the right kidney of mice by electroporation. Luciferase expression levels were measured to determine longevity of the expression.

RESULTS

Significantly high luciferase expression levels were observed in cultured RENCA, TCMK-1, and HEK293 cells over 1 month compared with controls (non-integrase system). The luciferase cDNA sequence was integrated at a pseudo attP site termed mpsL1. In vivo luciferase expression levels in the integrase group were sustained and significantly higher than those in the control group over 2 months. Furthermore, φC31 integrase-transfected cells had less genomic DNA damage caused by integrase expression.

CONCLUSIONS

These results demonstrated that the φC31 integrase system could produce long-term (2 months) in vivo gene expression in mouse kidney.

BACKGROUND

PhiC31 integrase is capable of conferring long-term transgene expression in various transfected tissues in vivo. In the present study, we investigated the activity of phiC31 integrase in mouse mammary glands.

METHODS

The normal mouse mammary epithelial cell line HC11 was transfected with FuGENE® HD Transfection Reagent (Roche Diagnostics, Shanghai, China). Transfection of the mouse mammary gland in vivo was performed by electrotransfer. Transgene expression was detected by western blotting and an enzyme-linked immunosorbent assay. Genomic integration and integration at mpsL1 was confirmed by a nested polymerase chain reaction.

RESULTS

An optimal electrotransfer protocol for the lactating mouse mammary gland was attained through investigation of different voltages and pulse durations. PhiC31 integrase mediated site-specific transgene integration in HC11 cells and the mouse mammary gland. In addition, the site-specific integration occurred efficiently at the ‘hot spot’ mpsL1. Co-delivery of PhiC31 integrase enhanced and prolonged transgene expression in the mouse mammary gland.

CONCLUSIONS

The results obtained in the present study show that the use of phiC31 integrase is a feasible and efficient method for high and stable transgene expression in the mouse mammary gland.