Adeno-associated virus encoding green fluorescent protein as a label for retinal pigment epithelium.
OBJECTIVE
To determine whether transduction with adeno-associated virus encoding green fluorescent protein (AAV-GFP) is useful for labeling transplanted retinal pigment epithelial cells (RPE).


METHODS
Transduction was performed by infection of confluent or subconfluent cultured feline RPE or by subretinal injection. Cells transduced in vitro were analyzed to determine label stability over time and label conservation with cell division. RPE transduced in vivo were harvested at 5 weeks for transplantation or immunohistochemical detection. Two cats received subretinal injections of harvested cells and were killed at 3 or 7 days.


RESULTS
In vitro transduction of confluent RPE resulted in stable GFP fluorescence for at least 3 months. There was a marked decline in fluorescence after cell division. Nonconfluent transduced cells conserved label after cell division but showed a marked decline in the number of cells, due to cell death. In vivo transduction resulted in a high level of labeling, allowing labeled cells to be harvested and transplanted. Transplanted cells were detected immunohistochemically. Photoreceptor labeling was detected over areas containing a high density of transplanted, labeled RPE derived from cells transduced in vivo. Possible light toxicity to transduced RPE was observed.


CONCLUSIONS
AAV-GFP-labeling of confluent cultured RPE and RPE in situ can be used to identify transplanted RPE, with some reservations. Cell division may cause dilution of the label, and release of cell contents into the subretinal space may cause label transfer to photoreceptors. Exposure to light of transduced cells should be limited.
Hansen
Kathleen A
KA
Institute of Ophthalmology and Visual Science, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 01701, USA.
Sugino
Ilene K
IK
Yagi
Fumihiko
F
Wang
Hao
H
Tsukahara
Itsuro
I
Gullapalli
Vamsi
V
Bennett
Jean
J
Zarbin
Marco A
MA
D000818:Animals; D002415:Cats; D017690:Cell Transplantation; D002478:Cells, Cultured; D000229:Dependovirus / Q000235:genetics*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism* / Q000637:transplantation; D012160:Retina / Q000473:pathology / Q000601:surgery; D013194:Staining and Labeling / Q000379:methods; D014161:Transduction, Genetic
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins