65 SCRIPTAID CORRECTS GENE EXPRESSION OF A FEW ABERRANTLY REPROGRAMMED TRANSCRIPTS IN NUCLEAR TRANSFER PIG BLASTOCYST STAGE EMBRYOS

Abstract

Scriptaid is a histone deacetylase inhibitor (HDACi) that can increase cloning efficiency. The objective of this study was to identify aberrantly reprogrammed transcripts by performing transcriptional profiling between in vivo (IVV), nuclear transfer (NT) blastocyst stage embryos and the donor cell line (cells). This was followed by measuring HDAC activity (Epigentek) in zygotes and by real-time PCR on a selected subset of genes at the blastocyst stage to determine if Scriptaid treatment (NTS) corrected the aberrant gene expression. NTS embryos were treated with 500 nM Scriptaid for 14 h after activation. NT and NTS embryos were transferred into gilts on Day 0 or 1 of oestrus and collected 6 days later by uterine flush. IVV samples were collected on Day 8 of gestation. 3 pools of 10 to 15 embryos and cells were collected for each treatment and analysed twice. For transcriptional profiling, total RNA was isolated by using Trizol (Invitrogen, Carlsbad, CA, USA), amplified by using an Ovation Ribo-SPIA linear amplification kit (Nugen), labelled with Cy5 and compared to reference labelled with Cy3. Lowess normalization and analysis was performed in Genespring 7.3.1. ANOVA was performed with the Benjamini and Hochberg False Discovery Rate. Transcripts that were different between IVV and NT (P ≤ 0.20) and significantly different from the donor cell line (P ≤ 0.05) were classified as being aberrantly reprogrammed. This comparison resulted in 119 under- and 60 over-compensated transcripts. Functional annotation classification was performed in DAVID and identified under-compensated pathways (oxidative phosphorylation and protein biosynthesis) and over-compensated pathways (chromatin packaging/remodelling and protein complex assembly). Fourteen transcripts were chosen for real-time PCR validation and evaluation of the effect of Scriptaid. Relative gene expression was compared between IVV, NT, NTS, and cells by the comparative Ct method with SYBR Green Supermix (Bio-Rad) and statistical analysis was performed in SAS 9.1 (SAS Institute Inc., Cary, NC, USA) by using a least significant difference test (P ≤ 0.05). NTS embryos had 3 transcripts returning to the same level as IVV (H3F3A, CAPG, and SEPT7). The level of the majority of the transcripts (8/14) was not affected by NTS treatment, e.g. histone deacetylase SIRT1 and H1 histone, member 0 (H1F0). However, Scriptaid treatment caused COX5A to be further over compensated in NTS with expression levels higher than IVV and NT. 2 transcripts had expression levels that were lower in NTS compared to both IVV and NT including GPD1L and EIF3E. Scriptaid treatment significantly affected gene expression in 6 of the 14 transcripts evaluated. Scriptaid treatment of the reconstructed zygotes did not affect the majority of the transcripts when measured at the blastocyst stage. HDAC activity was significantly reduced in NTS compared to NT 1-cell stage embryos (P ≤ 0.038). While Scriptaid reduced HDAC activity, it returned only a few genes to normal IVV levels. This project was supported in part by the USDA NRI (2006-35203-17282) and Food for the 21st Century.