Optimization of non-viral gene transfer to human primary retinal pigment epithelial cells.

PURPOSE

To optimise the high efficiency, non-viral transfer of DNA to retinal pigment epithelial (RPE) cells in vitro.

METHODS

A mammalian expression vector (pcDNA3.1) containing a firefly luciferase (luc) cDNA was used to transfect RPE cells using different chemical methods; calcium phosphate, DEAE-dextran and, liposomes-based transfection techniques. Transfection was optimised for both dose and time of exposure. The efficiency of gene transfer and cytotoxicity was measured 48 hours post-transfection using luciferase and MTT assays, respectively. The percentage of transfected cells (using optimal conditions) was determined with a construct expressing a jellyfish green fluorescent protein (GFP) using flow cytometery.

RESULTS

Calcium phosphate and DEAE-dextran techniques failed to transfect the vector and led to high cytotoxicity. Liposomes-based methods successfully transferred the vector to RPE cells, but the efficiency varied for different liposomes; Tfx-50 > Lipofectin > Lipofectamine > Cellfectin > DMRIE-C. No significant cytotoxicity was observed with any of the liposome treatments. Optimal transfection was achieved with Tfx-50 at a 3:1 ratio of DNA:liposome; between 12-15% of cells being transfected.

CONCLUSIONS

Efficient and non-toxic transfer of functional genes into primary RPE cells in vitro can be successfuly achieved by liposomes-based techniques. Tfx-50 appears to be a promising non-viral vector for RPE gene transfer.