Determination of EPAC2 function using EPAC2 null Min6 sublines generated through CRISPR-Cas9 technology.

摘要: Abstract Min6 cells, a mouse β cell line derived from transgenic expressing the large T-antigen of SV40 in pancreatic beta are commonly utilized as an in vitro cellular model for investigating targets involved insulin secretion. Epac2, exchange protein that can be directly activated by cyclic AMP (cAMP), is critical pharmacologic stimuli-induced secretion and has been hypothesized to direct target sulfonylurea. Previous loss function studies only specifically knocked out EPAC2 isoform A, leaving other two isoforms intact. In this study, we investigated cells generating knock-out sublines using CRISPR-Cas9 technology, removing all three EPAC2. Our results indicate successfully cloned single after electroporation with plasmids specific guide RNA, Cas9 GFP, followed sorting GFP cells. Two clones were found have nucleotide deletion targeted site gene sequencing, therefore creating frame shift exon 13. The null unexpectedly increased at basal level elevated total intracellular content. However, deficiency impaires glucose sulfonylurea induced without affecting binding Potassium chloride remains Interestingly, cAMP levels remained unchanged during these processes. To understand global EPAC2, RNA Seq study was performed, which reveals affects expression multiple previously unrecognized genes, suggesting through pathways addition being sensor.