Ετικέτες

Τετάρτη 31 Ιανουαρίου 2018

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

S03037207.gif

Publication date: Available online 31 January 2018
Source:Molecular and Cellular Endocrinology
Author(s): Haiyan Xu, Yi Yang, Yiping Chen, Uwe Muller, Sharanya Iyer, Jeremy Presland, Ruojing Yang, Ilona Kariv
Min6 cells, a mouse β cell line derived from transgenic mouse expressing the large T-antigen of SV40 in pancreatic beta cells, are commonly utilized as an in vitro cellular model for investigating targets involved in insulin secretion. Epac2, an exchange protein that can be directly activated by cyclic AMP (cAMP), is critical for pharmacologic stimuli-induced insulin secretion and has been hypothesized to be a direct target of sulfonylurea. Previous loss of function studies only specifically knocked out EPAC2 isoform A, leaving the other two isoforms intact. In this study, we investigated the function of EPAC2 in Min6 cells by generating EPAC2 knock-out sublines using CRISPR-Cas9 technology, by removing all three isoforms of EPAC2. Our results indicate that Min6 cells can be successfully cloned from a single cell after electroporation with plasmids expressing EPAC2 specific guide RNA, Cas9 and GFP, followed by sorting for GFP expressing single cells. Two clones were found to have a single nucleotide deletion in targeted site of EPAC2 gene by sequencing, therefore creating a frame shift in exon 13. The EPAC2 null clones unexpectedly have increased secretion of insulin at basal level and elevated total intracellular insulin content. However, EPAC2 deficiency impaired glucose and sulfonylurea induced insulin secretion without affecting sulfonylurea binding to cells. Potassium chloride induced insulin secretion remains intact. Interestingly, cAMP levels remained unchanged in EPAC2 null cells during these processes. To understand the global function of EPAC2, RNA Seq study was performed, which reveals that EPAC2 deficiency affects expression of multiple previously unrecognized genes, suggesting that EPAC2 can function through multiple pathways in addition to being a cAMP sensor.



http://ift.tt/2rWAL9c

Δεν υπάρχουν σχόλια:

Δημοσίευση σχολίου

Αναζήτηση αυτού του ιστολογίου