Endotoxin-free preparations of plasmid DNA were prepared using the GenElute TM Endotoxin free plasmid midi prep kit (Sigma). Crandall-Reese Feline Kidney cells (CRFKs) were seeded into a 12 well tray at approximately 40-50% confluence. CRFK cells were transfected with no DNA (negative control), pSV-b-Gal (control for b-galactosidase activity), pEGFP-C1/JV 59 GS/lacZ and pEGFP-C1/IRES/lacZ (control for IRES activity) using the Superfect TM transfection reagent (Qiagen) as per the manufacturer's recommendations. Following a 16 hour incubation the cells were observed for EGFP expression using a Leica Leitz DMRB fluorescence microscope. The cells were washed in PBS and fixed using a 0.5% solution of glutaraldehyde for 30 min at room temperature. The cells were incubated with an X-Gal stain solution: 5 mM K 3 Fe(CN) 6 , 5 mM K 4 Fe(CN) 6 , 2 mM MgCl 2 , 1x X-Gal (Sigma) for 4 hours at 37uC and were observed for b-glactosidase activity by light microscopy. The experiment was performed more than once to confirm the results.