RNA-seq analysis of Myt3 suppression and over-expression in mouse pancreatic islets

Purpose: Myt3 is a putative pro-survival factor in pancreatic islets and is expressed from E18.5 onwards. The genes that Myt3 regulates and its role in islet-cell development, function and survival are not fully known. The purpose of this study was to determine the changes in gene expression following the suppression or over-expression of Myt3 to identify possible pathways that may be regulated by Myt3. Methods: Pancreatic islets were isolated from both male and female C57/B6 mice by collagenase digestion and were picked by hand. Hand-picked islets were plated onto 804G, a complete extracellular-matrix (ECM) produced by a rat bladder carcinoma cell line, treated tissue culture dishes and transduced with 1x106 pfu virus overnight. The next day media was changed to fresh RPMI and islets were cultured for 7 days. At the end of the culture period islets were dispersed and FACs sorted for GFP+ cells. Sorted cells from at least 5 experiments were pooled and RNA was isolated using Trizol reagent. 800ng of RNA was used in the Illumina TruSeq kit for library preparation. Results: Comparison of gene expression levels between control-transduced islets and either shMyt3- or Myt3-transduced islets resulted in the identification of 51 and 89 significantly altered genes respectively. Conclusions: Our study represents the first comprehensive analysis of genes that are potentially regulated by Myt3 and highlight potential pathways that are likely important for mediating Myt3''s effects of islet development, function and survival. Overall design: Isolated islets were transduced with adenoviruses, cultured for 7 days on ECM and FACS sorted. Cells from up to 5 experiments were pooled and sequenced.