Mouse retinal pigmented epithelial cell lines retain their phenotypic characteristics after transfection with human papilloma virus: a new tool to further the study of RPE biology.
Development of immortalized mouse retinal pigmented epithelial cell (RPE) lines that retain many of their in vivo phenotypic characteristics, would aid in studies of ocular diseases including age related macular degeneration (AMD). RPE cells were isolated from 18-month-old (estrogen receptor knockout) ERKOalpha and ERKObeta mice and their C57Bl/6 wildtype littermates. RPE65 and cellular retinaldehyde binding protein (CRALBP) expression, in vivo markers of RPE cells, were detected by real-time RT-PCR and western analysis. We confirmed the presence of epithelial cell markers, ZO1, cytokeratin 8 and 18 by immunofluorescence staining. In addition, we confirmed the distribution of actin filaments and the expression of ezrin. To develop cell lines, RPE cells were isolated, propagated and immortalized using human papilloma virus (HPV) 16 (E6/E7). RPE-specific markers and morphology were assessed before and after immortalization. In wildtype littermate controls, there was no evidence of any alterations in the parameters that we examined including MMP-2, TIMP-2, collagen type IV, and estrogen receptor (ER)alpha and ERbeta protein expression and ER copy number ratio. Therefore, immortalized mouse RPE cell lines that retain their in vivo phenotype can be isolated from either pharmacologically or genetically manipulated mice, and may be used to study RPE cell biology.
Catanuto
Paola
P
Department of Surgery, University of Miami, Miller School of Medicine, Miami, Florida 33136, USA. pcatanuto@med.miami.edu
Espinosa-Heidmann
Diego
D
Pereira-Simon
Simone
S
Sanchez
Patricia
P
Salas
Pedro
P
Hernandez
Eleut
E
Cousins
Scott W
SW
Elliot
Sharon J
SJ
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002455:Cell Division / Q000502:physiology; D002461:Cell Line, Transformed; D016764:Cell Polarity / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D002472:Cell Transformation, Viral; D005136:Eye Proteins / Q000378:metabolism; D005260:Female; D052162:Human papillomavirus 16; D007365:Intercellular Junctions / Q000378:metabolism / Q000648:ultrastructure; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010641:Phenotype; D011960:Receptors, Estrogen / Q000172:deficiency / Q000235:genetics; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014162:Transfection; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase
D002352:Carrier Proteins; D005136:Eye Proteins; D011960:Receptors, Estrogen; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C020932:11-cis-retinal-binding protein