Adeno-Associated Virus Encoding Green Fluorescent Protein as a Label for Retinal Pigment Epithelium

• Published in: Investigative ophthalmology & visual science Vol. 44; no. 2; pp. 772 - 780
• Main Authors: Hansen, Kathleen A, Sugino, Ilene K, Yagi, Fumihiko, Wang, Hao, Tsukahara, Itsuro, Gullapalli, Vamsi, Bennett, Jean, Zarbin, Marco A
• Format: Journal Article
• Language: English
• Published: Rockville, MD ARVO 01-02-2003; Association for Research in Vision and Ophtalmology
• Subjects: Animals; Biological and medical sciences; Cats; Cell Transplantation; Cells, Cultured; Dependovirus - genetics; Genetic Vectors; Green Fluorescent Proteins; Indicators and Reagents - metabolism; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Medical sciences; Pigment Epithelium of Eye - cytology; Pigment Epithelium of Eye - metabolism; Pigment Epithelium of Eye - transplantation; Retina - pathology; Retina - surgery; Staining and Labeling - methods; Surgery (general aspects). Transplantations, organ and tissue grafts. Graft diseases; Surgery of the eye and orbit; Transduction, Genetic; Fissipedia; Cell culture; Carnivora; Adenoviridae; Pigment epithelium; Homograft; Labelling; Retina; Identification; Experimental study; Virus; Vertebrata; Mammalia; Surgery; Human adenovirus; Animal; Cat; Green fluorescent protein; Graft; Mastadenovirus; Transduction
• ISSN: 0146-0404; 1552-5783; 1552-5783
• DOI: 10.1167/iovs.02-0091

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