Animal Reproduction Science 141 (2013) 164–171 Contents lists available at ScienceDirect Animal Reproduction Science journal h om epa ge: www.elsevier.com/locate/anireprosci Treatment of fetal fibroblasts with DNA methylation inhibitors and/or histone deacetylase inhibitors improves the development of porcine nuclear transfer-derived embryos Yun-Fei Diao, Ken-Ji Naruse, Rong-Xun Han, Xiao-Xia Li, Reza K. Oqani, Tao Lin, Dong-Il Jin ∗ Department of Animal Science & Biotechnology, Chungnam National University, Daejeon City 338-708, Republic of Korea a r t i c l e i n f o Article history: Received 25 March 2013 Received in revised form 9 August 2013 Accepted 13 August 2013 Available online 22 August 2013 Keywords: Porcine nuclear transfer Epigenetic modification 5-aza-dC TSA Embryo development a b s t r a c t This study investigated whether treating fetal fibroblast cells (donor cells) with epigenetic modification-inducing drugs could improve the development of porcine cloned embryos. Donor cells were treated with different DNA methylation inhibitors (5-aza-dC, zebularine or RG108; 5 nM) or histone deacetylase inhibitors (TSA, NaBu or SCR; 50 nM) for 1 h, and then subjected to SCNT. All of the treated groups showed significantly higher blastocyst formation rates compared to the control group. We chose 5-aza-dC and TSA as a combined treatment, and found that donor cells co-treated with 2.5 nM 5-aza-dC for 1 h and subse- quently treated with 50 nM TSA for another 1 h before SCNT showed significantly improved blastocyst rates compared to the control, 5-aza-dC-treated, and TSA-treated groups. The levels of DNA methylation were decreased (though not to a significant degree) in donor cells treated with 5-aza-dC, TSA or both. The histone H3 acetylation levels were signif- icantly increased in donor cells treated with TSA or co-treated with 5-aza-dC and TSA. Donor cells simultaneously co-treated with 5 nM 5-aza-dC and 50 nM TSA for 1 h showed increased apoptosis of SCNT blastocysts. However, when we decreased the concentration of 5-aza-dC to 2.5 nM, the co-treatment induced less apoptosis among SCNT blastocysts and the blastocyst development rate improved. Together, these results indicate that treatment of donor cells with 5-aza-dC, TSA, or TSA plus a low dose of 5-aza-dC could improve the blastocyst development of porcine cloned embryos. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Cloning via somatic cell nuclear transfer (SCNT) has been successfully performed in various species (Wakayama, 2007; Campbell et al., 2007; Kishigami et al., 2008). The first successful cloning of a pig was reported in 2000 (Onishi et al., 2000; Polejaeva et al., 2000). How- ever, the cloning of live pigs has proven inefficient, partially due to poor development of nuclear-transfer (NT) embryos to blastocysts (Pratt et al., 2006). Inefficient nuclear ∗ Corresponding author. Tel.: +82 42 821 5876; fax: +82 42 822 6712. E-mail address: dijin@cnu.ac.kr (D.-I. Jin). reprogramming is thought to be a major cause of abnor- mal gene expression patterns and the inefficiency of SCNT (Niemann and Wrenzycki, 2000; Tanaka et al., 2001; Rideout et al., 2001). Aberrations in epigenetic processes, such as DNA methylation and histone acetylation, can lead to abnormal gene expression (Dannenberg and Edenberg, 2006). Epigenetic modification-inducing chemicals, including DNA methylation inhibitors and histone deacetylase inhibitors (HDACIs), have been used for the epigenetic modification of somatic cells. The DNA methylation inhibitors, which decrease methylation, include 5-aza-2 ′ - deoxyctidine (5-aza-dC), RG108 [2-(1,3-dioxo-1,3- dihydro-2H-isoindol-2-yl)-3- (1H-indol-3-yl) propanoic 0378-4320/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anireprosci.2013.08.008