Purchase this article with an account.
Knut Stieger, Franziska Wagner, Brigitte Mueller; Analysis of the DNA repair machinery status in the retina, other tissues and standard cell lines. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1151.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The majority of therapeutic genome editing studies targeting retinal dystrophies employ ex vivo approaches in induced pluripotent stem cells. In contrast, the idea of correcting the disease causing mutations in vivo in the retina has many advantages. However, the state of the DNA repair machinery in post-mitotic retinal cells is largely unknown. Especially in the retina, the DNA double strand break repair is considered to be of low activity. The aim of this study was to compare gene expression levels of proteins involved in DNA repair in different tissues and different cell lines in order to assess the suitability of cell lines as model systems for in vivo approaches.
Gene expression levels of proteins involved in DNA damage sensing, homology directed repair (HR), non-homologous endjoining (NHEJ) or micro-homology mediated end joining (MMEJ) were assessed in 3 months old retinae of wild-type mice and retinae of an X-linked retinitis pigmentosa mouse model (B6.129-Rpgrtm1Sti), as well as in liver, kidney, brain, lung, and gonads of both mouse lines. Cell lines used in this study are the C2C12 (murine) and HEK293T (human) lines. Tissue was processed to obtain RNA, and cDNA following RT-PCR. Quantitative PCR (Eppendorf Realplex S4) was performed for genes like 53bp1, CtIP, DNA-Pkc, Ku80, BRCA1 and normalized to GAPDH. Relative quantification was calculated using the ddCt method.
Most of the analysed genes were similarly expressed in retina and other tissues. However, compared to cell cultures, gene expression levels varied dramatically. The most prominent differences were seen for Ku80, the expression of which was more than 300fold lower in cell cultures compared to tissue, and 53bp1, the expression of which was more than 400 fold higher in the retina. BRCA1 and CtIP expression levels were moderately higher (8-25 fold) in cell lines compared to the retina. DNA-Pkc expression levels were quite low and did not differ considerably between tissues and cell lines.
The DNA repair machinery seems to be in a similar state in the retina and other tissues, but fundamentally different between tissues and cell lines. Since this likely due to the post-mitotic status in tissue compared to the mitotic status in cell lines, the latter may not be the optimal model system to develop genome editing as treatment strategy in vivo.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
This PDF is available to Subscribers Only