P15 Identification of pro‐apoptotic signalling through the GPRC5B receptor in beta cells by CRISPR‐Cas9‐mediated genome editingP ATANES, I Ruz‐Maldonado, R Hawkes, B Liu, S Amisten, SJ PersaudDepartment of Diabetes, Faculty of Life Sciences & Medicine, King's College London, London, UKBackground/AimsGPRC5B is an orphan receptor that is highly expressed in human and mouse islets, and we have previously reported that its down‐regulation in islets protects against apoptosis. As there are no agonists identified for GPRC5B, we investigated its role in MIN6 beta cells by stably deleting it using the CRISPR‐Cas9 technology, then re‐introducing it in a graded fashion.Methods: Efficiency of GFP plasmid delivery into MIN6 beta cells was determined by cell sorting following transfection with eight reagents. CRISPR‐Cas9 was used to stably delete the Gprc5b gene in MIN6 beta cells, and 0.01% to 100% Gprc5b plasmid was introduced by transient transfection to restore Gprc5b expression. Standard techniques were used to quantify Gprc5b mRNA expression, apoptosis, and signalling pathways downstream of GPRC5B were identified using Cignal 45‐Pathway Reporter Arrays and western blotting.Results: JetPRIME® provided the best MIN6 beta cell transfection efficiency (29 ± 0.5% GFP‐positive cells). Isolation of Gprc5b KO cell clones using CRISPR‐Cas9 was established, and DNA sequencing confirmed deletion of Gprc5b
Diabetic Medicine – Wiley
Published: Jan 1, 2018
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