High efficiency non-viral transfection of retinal and iris pigment epithelial cells with pigment epithelium-derived factor.
Transplantation of pigment epithelial cells in patients with age-related macular degeneration and Parkinson's disease has the potential to improve functional rehabilitation. Genetic modification of cells before transplantation may allow the delivery of neuroprotective factors to achieve functional improvement. As transplantation of cells modified using viral vectors is complicated by the possible dissemination of viral particles and severe immune reactions, we have explored non-viral methods to insert genetic material in pigment epithelial cells. Using lipofection or nucleofection ARPE-19 cells, freshly isolated and primary retinal and iris pigment epithelial (IPE) cells were transfected with plasmids encoding green fluorescent protein (GFP) and with three plasmids encoding recombinant pigment epithelium-derived factor (PEDF) and GFP. Transfection efficiency was evaluated by fluorescence microscopy and stability of protein expression by immunoblotting. Pigment epithelial cells were successfully transfected with plasmid encoding GFP. Expression of GFP in ARPE-19 was transient, but was observed for up to 1 year in IPE cells. Analysis of pigment epithelial cells transfected with PEDF plasmids revealed that PEDF fusion proteins were successfully expressed and functionally active. In conclusion, efficient transfer of genetic information in pigment epithelial cells can be achieved using non-viral transfection protocols.
Thumann
G
G
IZKF Biomat, RWTH Aachen University, Aachen, Germany. GThumann@ukaachen.de
Stöcker
M
M
Maltusch
C
C
Salz
A K
AK
Barth
S
S
Walter
P
P
Johnen
S
S
D000818:Animals; D002478:Cells, Cultured; D018274:Electroporation; D005136:Eye Proteins / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D007498:Iris / Q000378:metabolism*; D009414:Nerve Growth Factors / Q000235:genetics*; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D010957:Plasmids; D051381:Rats; D015843:Serpins / Q000235:genetics*; D013552:Swine; D014162:Transfection / Q000379:methods*; D000099174:Pigment Epithelium-Derived Factor
D005136:Eye Proteins; D049452:Green Fluorescent Proteins; D009414:Nerve Growth Factors; D015843:Serpins; D000099174:Pigment Epithelium-Derived Factor