Transcriptomic profiling of untreated and oxidative stress treated Wild-type and IPO13-/- mouse embryonic stem cells to study the role of IPO13 in transcriptional response to stress.

Purpose: The importin (IPO) superfamily member IPO13 is able to mediate nuclear transport bidirectionally. To better understand IPO13 involvement in cellular signalling, we performed transcriptomic analysis on wild-type and IPO13-knockout mouse embryonic stem cells (mESCs), with gene ontology (GO) annotation results indicating enrichment of differentially expressed genes involved in stress responses and regulation of apoptosis. To examine IPO13's contribtion to the response to cellular stress, we employed RNA sequencing to investigate and compare the transcription profile of wild-type and IPO13-knockout ESCs in the presence and absence of oxidative stress. Methods: Wild-type and IPO13-knock out mESCs were treated with or without 125 µM H2O2 for 1 h, after which the medium was replaced and cells recovered in the absence of H2O2 for 2 h. Total RNA was isolated and used to generate sequencing libraries using Illumina TruSeq Stranded mRNA Library Prep Kit. Sequencing was then performed on an Illumina HiSeq2500 platform. qRT-PCR validation was performed using SYBR Green assays. Results: Results comparing Wild-type and IPO13-knock out cell lines indicated that IPO13 knock out results in the down-regulation of 338 genes and up-regulation of 536 genes. This included genes involved in stress response and apoptotic pathways. Comparison of specific gene subset differentially expressed in response to H2O2 treatment identifiedd 277 IPO13-dependent genes up- or down-regulated exclusively in the Wild-type cell line and not in the IPO13-knock out cell line, highlighting the importance of IPO13 in the transcriptional response to oxidative stress. Conclusion: IPO13 plays a key role in the stress response. Examination of mRNA levels from Wild-type and IPO13-/- mouse embryonic stem cells under untreated and hydrogen peroxide treated conditions to identify genetic targets of IPO13 in cells undergoing oxiative stress.