Derivation and characterization of putative embryonic stem cells from cloned rabbit embryos Payungsuk Intawicha a, b , Chawalit Siriboon a, c , Chien-Hong Chen d , Yung-Tsung Chiu a, e , Tzu-An Lin a , Michel Kere a, f , Neng-Wen Lo g , Kun-Hsiung Lee d , Li-Yung Chang c, h , Hsing-I. Chiang a , Jyh-Cherng Ju a, c, h, i, * a Department of Animal Science, National Chung Hsing University, Taichung, Taiwan, Republic of China b Division of Animal Science, School of Agriculture and Natural Resources, University of Phayao, Phayao, Thailand c Department of Animal Science, Faculty of Agriculture, Ubon Ratchathani University, Ubon Ratchathani, Thailand d Division of Biotechnology, Agriculture Technology Research Institute, Hsinchu City, Taiwan, Republic of China e Department of Medical Research, Taichung Veterans General Hospital, Taichung, Taiwan, Republic of China f Departement d’Elevage, Institut du Developpement Rural, Universite Polytechnique de Bobo, Bobo Dioulasso, Burkina Faso g Department of Animal Science and Biotechnology, Tunghai University, Taichung, Taiwan, Republic of China h Department of Biomedical Informatics and Medical Engineering, Asia University, Taichung, Taiwan, Republic of China i Core Laboratory for Stem Cell Research, Medical Research Department, China Medical University Hospital, Taichung, Taiwan, Republic of China article info Article history: Received 1 November 2015 Received in revised form 24 May 2016 Accepted 27 May 2016 Keywords: Somatic cell nuclear transfer Embryonic stem cells Embryoid bodies Rabbit ES cells abstract The present study aimed to establish embryonic stem (ES) cell lines, i.e., ntES cells, using rabbit blastocyst stage embryos cloned by somatic cell nuclear transfer. First, we investi- gated the development of cloned rabbit embryos reconstructed with normal fibroblasts and fibroblasts transfected with enhanced green fluorescence protein (eGFP). Blastocyst rates were 27.4% and 23.9%, respectively, for the embryos reconstructed with normal fibroblasts and fibroblasts transfected with eGFP compared with that from the parthe- nogenetic group (43.1%). One ntES cell line was established from embryos reconstructed with eGFP-transfected fibroblasts (1 of 17, 5.9%), and three ntES cell lines were derived from those with normal fibroblasts (3 of 17,17.6%). All the ntES cell lines retained alkaline phosphatase activity and expressed ES cell–specific markers SSEA-4, Oct-4, TRA-1-60, and TRA-1-81. The pluripotency was further confirmed by reverse transcription–polymerase chain reaction analyses of Oct-4, Nanog, and Sox-2 expressions in ntES cell lines. The differentiation capacity of ntES cells was also examined in vitro and in vivo, by which these ntES cell lines were able to differentiate into all three germ layers through embryoid bodies and teratomas. In conclusion, it is apparent that the efficiency of ntES cells derived using eGFP-transfected donor cells is lower than that with nontransfected, normal fibro- blasts donor cells. Similar to those from parthenogenetic embryos, all ntES cell lines derived from cloned rabbit embryos are able to express pluripotency markers and retain their capability to differentiate into various cell lineages both in vitro and in vivo. Ó 2016 Elsevier Inc. All rights reserved. 1. Introduction Rabbits (Oryctolagus cuniculus) are classical laboratory animals with many advantages over rodents and other species and have been used for studying human diseases including hypertension [1,2], myocardial infarction [3–5], The first two authors contributed equally to this article. * Corresponding author. Tel.: þ886-4-2233-7203; fax: þ886-4-2233- 3641. E-mail address: jcju@dragon.nchu.edu.tw (J.-C. Ju). Contents lists available at ScienceDirect Theriogenology journal homepage: www.theriojournal.com 0093-691X/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2016.05.035 Theriogenology xxx (2016) 1–12