We developed an in vivo electroporation method to introduce foreign genes into retinal ganglion cells (RGCs). After the intravitreous injection of the plasmid gene (20 μg), five electric pulses (6 V/cm, 100 ms duration) were each delivered twice with 5 min interval to the rat eye using a contact lens-type electrode (cathodal) attached to the cornea and a needle electrode (anodal) inserted to the middle of the forehead. The efficiency of the genetic introduction into RGCs and tissue damage to the eyeball was evaluated using a green fluorescent protein (GFP) gene, TUNEL and histological observation. Dil retrograde labeling revealed that 24.4 ± 4.7% of all RGCs were electrointroduced with the GFP gene. TUNEL and histological analysis showed a few tissue damages in the cornea, lens and retina. To confirm whether this method can actually rescue damaged RGCs, glial cell line-derived neurotrophic factor (GDNF) was electrointroduced into RGCs after optic nerve transection. After the electrointroduction, a significant increase in the number of surviving RGCs was observed 2 and 4 weeks after the optic nerve transection, and the decrease of caspase 3 and 9 was detected by RT-PCR. These results suggest that this method may be useful for the delivery of genes into RGCs with simplicity and minimal tissue damage.
- Glial cell line-derived neurotrophic factor (GDNF)
- In vivo gene therapy
ASJC Scopus subject areas
- Molecular Medicine
- Molecular Biology