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J Biol Chem. 2016 Jul 29;291(31):16339-47. doi: 10.1074/jbc.M116.729467. Epub 2016 May 31.

The Clustered, Regularly Interspaced, Short Palindromic Repeats-associated Endonuclease 9 (CRISPR/Cas9)-created MDM2 T309G Mutation Enhances Vitreous-induced Expression of MDM2 and Proliferation and Survival of Cells.

Author information

1
From the Schepens Eye Research Institute, Massachusetts Eye and Ear, and Departments of Ophthalmology and the Shanxi Eye Hospital, Taiyuan, Shanxi 030000, China.
2
From the Schepens Eye Research Institute, Massachusetts Eye and Ear, and Departments of Ophthalmology and.
3
From the Schepens Eye Research Institute, Massachusetts Eye and Ear, and Departments of Ophthalmology and Pathology, Harvard Medical School, Boston, Massachusetts 02114.
4
the Broad Institute of the Massachusetts Institute of Technology and Harvard University, Cambridge, Massachusetts 02142, and.
5
From the Schepens Eye Research Institute, Massachusetts Eye and Ear, and Departments of Ophthalmology and Hetian_lei@meei.harvard.edu.

Abstract

The G309 allele of SNPs in the mouse double minute (MDM2) promoter locus is associated with a higher risk of cancer and proliferative vitreoretinopathy (PVR), but whether SNP G309 contributes to the pathogenesis of PVR is to date unknown. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas) 9 from Streptococcus pyogenes (SpCas9) can be harnessed to manipulate a single or multiple nucleotides in mammalian cells. Here we delivered SpCas9 and guide RNAs using dual adeno-associated virus-derived vectors to target the MDM2 genomic locus together with a homologous repair template for creating the mutation of MDM2 T309G in human primary retinal pigment epithelial (hPRPE) cells whose genotype is MDM2 T309T. The next-generation sequencing results indicated that there was 42.51% MDM2 G309 in the edited hPRPE cells using adeno-associated viral CRISPR/Cas9. Our data showed that vitreous induced an increase in MDM2 and subsequent attenuation of p53 expression in MDM2 T309G hPRPE cells. Furthermore, our experimental results demonstrated that MDM2 T309G in hPRPE cells enhanced vitreous-induced cell proliferation and survival, suggesting that this SNP contributes to the pathogenesis of PVR.

KEYWORDS:

CRISPR/Cas9, MDM2T309G; ERK; apoptosis; cell growth; epithelial cell; genetic polymorphism; molecular biology

PMID:
27246850
PMCID:
PMC4965581
DOI:
10.1074/jbc.M116.729467
[Indexed for MEDLINE]
Free PMC Article

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