TY - JOUR
T1 - Mouse retinal pigmented epithelial cell lines retain their phenotypic characteristics after transfection with human papilloma virus
T2 - A new tool to further the study of RPE biology
AU - Catanuto, Paola
AU - Espinosa-Heidmann, Diego
AU - Pereira-Simon, Simone
AU - Sanchez, Patricia
AU - Salas, Pedro
AU - Hernandez, Eleut
AU - Cousins, Scott W.
AU - Elliot, Sharon J.
N1 - Funding Information:
This work was supported in part by National Institutes of Health National Eye Institute Grant RO1 EY14477-04 (SJE and SWC). We would like to thank Hong Yu for the gift of HPV and Jason Fritz for technical assistance with the confocal microscopy.
PY - 2009/1/5
Y1 - 2009/1/5
N2 - 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) ERKOα and ERKOβ 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)α and ERβ 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.
AB - 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) ERKOα and ERKOβ 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)α and ERβ 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.
KW - estrogen receptors
KW - extracellular matrix
KW - immortalization
KW - mouse retinal pigmented epithelial cells
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UR - http://www.scopus.com/inward/citedby.url?scp=57549114246&partnerID=8YFLogxK
U2 - 10.1016/j.exer.2008.10.013
DO - 10.1016/j.exer.2008.10.013
M3 - Article
C2 - 19013153
AN - SCOPUS:57549114246
SN - 0014-4835
VL - 88
SP - 99
EP - 105
JO - Experimental eye research
JF - Experimental eye research
IS - 1
ER -
