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Updated: Feb 20, 2026
Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells
Published on: February 10, 2021
Introduction of the MDM2 T309G Mutation in Primary Human Retinal Epithelial Cells Enhances Experimental Proliferative
Guohong Zhou1,2, Yajiang Duan1,2, Gaoen Ma1,3
1Schepens Eye Research Institute of Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, United States.
Purpose:
The murine double minute (MDM)2 is a critical negative regulator of the p53 tumor suppressor, and MDM2 SNP309G is associated with a higher risk of proliferative vitreoretinopathy (PVR); in addition, the MDM2 T309G created using clustered regularly interspaced short palindromic repeats (CRISPR)/associated endonuclease (Cas)9 enhances normal rabbit vitreous-induced expression of MDM2 and survival of primary human retinal pigment epithelial (hRPE) cells in vitro. The goal of this study was to determine whether this MDM2 T309G contributes to the development of experimental PVR.
Methods:
hRPE cells expressing MDM2 T309G or T309T only were treated with vitreous from human PVR donors (HV). The expression of MDM2 and p53 in the treated cells was examined by Western blot. The in vitro vitreous-induced cellular responses, such as contraction were assessed, and PVR was induced by intravitreal injection of the hRPE cells with MDM2 T309G or T309T only into rabbit eyes.
Results:
Western blot analyses indicated that treatment of hRPE cells with HV led to a significant increase (1.7 ± 0.2-fold) in the expression of MDM2 and a significant decrease in p53 in the cells expressing MDM2 T309G compared with those with MDM2 T309T. In addition, HV promoted contraction of the hRPE cells expressing MDM2 T309G significantly more than those with MDM2 T309T only. Furthermore, MDM2 T309G in the hRPE cells enhanced the development of PVR in a rabbit model.
Conclusions:
The MDM2 SNP309 in RPE cells enhances their potential of PVR pathogenesis.
Insights
The MDM2 SNP309 genetic variant in retinal pigment epithelial cells promotes proliferative vitreoretinopathy (PVR) development. This variant increases MDM2 expression and cellular contraction, contributing to PVR pathogenesis.
Area of Science:
- Ophthalmology
- Genetics
- Molecular Biology
Background:
- Murine double minute 2 (MDM2) is a key negative regulator of the p53 tumor suppressor protein.
- The MDM2 SNP309G variant is linked to an increased risk of proliferative vitreoretinopathy (PVR).
- Engineered MDM2 T309G variant enhances MDM2 expression and retinal pigment epithelial cell survival.
Purpose of the Study:
- To investigate the role of the MDM2 T309G variant in the pathogenesis of experimental PVR.
- To determine if MDM2 T309G influences MDM2 expression and cellular behavior in response to PVR factors.
Main Methods:
- Human retinal pigment epithelial (hRPE) cells with MDM2 T309G or T309T genotypes were treated with human PVR vitreous (HV).
- MDM2 and p53 expression levels were analyzed using Western blot.
- In vitro cellular contraction and in vivo PVR development in a rabbit model were assessed.
Main Results:
- HV treatment significantly increased MDM2 expression and decreased p53 levels in hRPE cells with the MDM2 T309G variant compared to T309T.
- hRPE cells expressing MDM2 T309G showed significantly greater contraction when treated with HV.
- The MDM2 T309G variant in hRPE cells exacerbated PVR development in the rabbit model.
Conclusions:
- The MDM2 SNP309 variant in RPE cells contributes to PVR pathogenesis.
- Elevated MDM2 expression due to SNP309 may enhance cellular responses that drive PVR.
- Targeting MDM2 or its regulatory pathways could be a therapeutic strategy for PVR.
