Purchase this article with an account.
Brigitte Muller, Franziska Wagner, Jessica Grebe, Knut Stieger; Time course of DNA damage response gene expression in murine retinal explant cultures. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5414.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The purpose of this study was to characterize the organ culture of murine neuroretina to establish gene expression of a focused panel of genes related to DNA damage signaling.
Neuroretina explants were prepared from wild type C57Bl6 mice and evaluated after 0.5 to 8 days in culture. Fresh retinae were used as controls. Explants were cultured in culture plate inserts with the photoreceptor layer facing the supporting PCTE-membrane. Eventually total RNA was isolated from explants and processed with a mouse DNA Damage Signaling Pathway RT² Profiler™ PCR Array. The genes featured are those associated with the ATR/ATM signaling network and transcriptional targets of DNA damage response. Using real-time PCR and the 2^ΔΔCT method we analyzed the relative gene expression
Gene expression of many genes associated with the ATR/ATM signaling, DNA double strand repair (DSB), apoptosis, and cell cycle are considerably down regulated between two and tenfold in retinal explants. Key players in the DSB repair like ATM, Mre11a, Nibrin, and Rad50 are down regulated up to five-fold. However, gene expression for individual proteins like Cdkn1a (p21) and Ppp1r15a get up regulated by two to tenfold during culture, both are associated with apoptosis and cell cycle arrest. Cdc25a is up regulated as well by two to fivefold and competent to activate the G1/S cyclin-dependent kinases Cdk4 and Cdk2 by removing inhibitory phosphate groups and can also activate Cdc2 (Cdk1), the principal mitotic Cdk. In general, major changes in gene expression of retinal explants seem to happen during the first two days in culture.
Our preliminary gene expression results of different stages of retinal explants fit well with our histological findings, where apart from disruption and truncation of photoreceptor outer and inner segments only minor morphological changes like sprouting of bipolar cell dendrites into the outer plexiform layer and isolated apoptotic nuclei in the outer and inner nuclear layer were detectable. Exposing retinal explants cultures to conditions like hypoxia or CNTF incubation are promising next steps to further investigate gene expression in the DNA damage response. By this we hope to establish retinal explant cultures as a model for gene therapeutic treatment, which represents an ideal intermediate step between cell culture and animal experiments.
This PDF is available to Subscribers Only