Retinal explant cultures of adult mice as a model studying gene editing

Abstract

Purpose : The purpose of this study was to detect DNA-DSB-repair proteins in organ culture of adult murine neuroretina to establish it as a model for gene editing.

Methods : Neuroretina explants were prepared from wild type C57Bl6 mice and a XLRP mouse model (B6J.SV129-Rpgr^tm1stie) and evaluated after 1 to 8 days in culture by immunohistochemistry or qPCR. Fresh retinae were used as controls. Explants were cultured in culture plate inserts containing complete culture medium with the photoreceptor layer facing the supporting PCTE-membrane. Total RNA was isolated from explants and processed for qPCR for DNA-damage and DNA-repair proteins like 53BP1, Ku80, DNA-PKcs, BRCA1, and CtIP. For histological investigations, explants where harvested and conserved as wholemounts or prepared for cryo-sectioning. Subsequently, retinal explants were counterstained by immunohistochemical markers for DNA damage and repair proteins γH2AX, Ku80, 53BP1, DNA-PKcs, and BRCA1 and analyzed by confocal microscopy.

Results : During the first week in culture retinal explants gene expression of Ku80, a key player in NHEJ, is up regulated up to three fold during the first three days in retinal explant culture. Expression of 53BP1, a regulator protein between HDR and NHEJ repair pathways gets up-regulated for two days and then down-regulated by two-fold. Likewise BRCA1, which antagonizes 53BP1 chromatin interactions. DNA-PKcs and CtIP showed no consistent change of expression level during retinal explant culture. With above mentioned antisera we localized individual DNA-damage and DSB-repair proteins in retinal cells in culture complementing our gene expression results.

Conclusions : Our morphological results on culturing retinal explants, yet without any manipulations, were the first step to establish retinal explants for gene editing as an intermediate step between cell culture and animal experiments as previously show. The detection of DNA-damage and DNA-repair proteins by immunohistochemistry and gene expression lays the ground to define optimal settings which can subsequently be modified by external drug applications.

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

 

Vertical cryostat sections of retinal explant cultures from 3 month old C57Bl6 mice labeled by 53BP1 antibody. Immunopositive nuclei are found in the inner nuclear layer (INL) and the ganglion cell layer (GCL) only. At 10 days in culture only very few nuclei show 53BP1-immunoreactivity.