September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
The CRISPR/Cas9-created MDM2 T309G contributes to the vitreous-induced cellular responses intrinsic to proliferative vitreoretinopathy
Author Affiliations & Notes
  • Gaoen Ma
    schepens eye reseach insstitute, Boston, Massachusetts, United States
  • Yajian Duan
    schepens eye reseach insstitute, Boston, Massachusetts, United States
  • Xionggao Huang
    schepens eye reseach insstitute, Boston, Massachusetts, United States
  • Patricia A D'Amore
    schepens eye reseach insstitute, Boston, Massachusetts, United States
  • Hetian Lei
    schepens eye reseach insstitute, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Gaoen Ma, None; Yajian Duan, None; Xionggao Huang, None; Patricia D'Amore, None; Hetian Lei, None
  • Footnotes
    Support  NIH R01 EY012509
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 5367. doi:
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      Gaoen Ma, Yajian Duan, Xionggao Huang, Patricia A D'Amore, Hetian Lei; The CRISPR/Cas9-created MDM2 T309G contributes to the vitreous-induced cellular responses intrinsic to proliferative vitreoretinopathy. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5367.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : The oncogene protein murine double minute 2(MDM2) is a key negative regulator of the p53 tumor suppressor. The G allele singe nucleotide polymorphisms (SNPs) (rs2279744) in the MDM2 promoter locus is associated with a higher risk of proliferative vitreoretinopathy (PVR). We used CRISPR/Cas9 to create the SNP G309 in the primary retinal pigment epithelial (PRPE) to determine whether the SNP G309 contributes to the pathogenesis of PVR.

Methods : For SpCas9 targets and generation of single guide (sg) RNAs, two 20-nucleotide targeted sequences were chosen preceding a 5’-NGG protospacer adjacent motif (PAM) sequence. Control sgRNA sequence was designed to target lacZ gene. The plasmids of pAAV-SpCas9D10A, pAAV-MDM2-sgRNA 1+2 (or pAAV-LacZ sgRNA) and a single strand homology directed repair (HDR) template together were transfected into PRPE cells by electroporation. The amplified DNA fragments around the designed mutation were subjected to Sanger DNA sequencing, surveyor nuclease assay and next generation sequencing. The expression of MDM2 and p53 in the modified and control cells was examined by western blot. Cellular responses to vitreous that are involved in PVR such as cell proliferation, contraction were assessed.

Results : Sanger DNA sequencing and surveyor nuclease assay demonstrated that the introduction of the MDM2 T309G SNP in the PRPE cells. Next generation sequencing indicated that there 42.52% MDM2 G309 and 57.19% MDM2 T309 in the PRPE cells transfected with pAAV-SpCas9D10A, pAAV-MDM2 sgRNA 1+2 plus the HDR template, but 100% MDM2 T309 in the lacZ sgRNA-transfected cells. Vitreous from experimental rabbits (RV) enhanced MDM2 expression(1.8±0.2), resulting a decrease(70±9%) in p53 in the PRPE cells with the MDM2 T309G compared to those with MDM2 T309T. Furthermore, RV promoted greater cell proliferation and contraction in the PRPE cells with the MDM2 T309G than those with MDM2 T309T.

Conclusions : The MDM2 T309G SNP in the PRPE cells leads to enhanced vitreous-induced expression of MDM2 and cellular responses involved to PVR, and may contribute to the pathogenesis of PVR.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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