title: An Improved System for Generation of Diploid Cloned Porcine Embryos Using Induced Pluripotent Stem Cells Synchronized to Metaphase.
abstract: Pigs provide outstanding models of human genetic diseases due to their striking similarities with human anatomy, physiology and genetics. Although transgenic pigs have been produced using genetically modified somatic cells and nuclear transfer (SCNT), the cloning efficiency was extremely low. Here, we report an improved method to produce diploid cloned embryos from porcine induced pluripotent stem cells (piPSCs), which were synchronized to the G2/M stage using a double blocking method with aphidicolin and nocodazole. The efficiency of this synchronization method on our piPSC lines was first tested. Then, we modified our traditional SCNT protocol to find a workable protocol. In particular, the removal of a 6DMAP treatment post-activation enhanced the extrusion rate of pseudo-second-polar bodies (p2PB) (81.3% vs. 15.8%, based on peak time, 4hpa). Moreover, an immediate activation method yielded significantly more blastocysts than delayed activation (31.3% vs. 16.0%, based on fused embryos). The immunofluorescent results confirmed the effect of the 6DMAP treatment removal, showing remarkable p2PB extrusion during a series of nuclear transfer procedures. The reconstructed embryos from metaphase piPSCs with our modified protocol demonstrated normal morphology at 2-cell, 4-cell and blastocyst stages and a high rate of normal karyotype. This study demonstrated a new and efficient way to produce viable cloned embryos from piPSCs when synchronized to the G2/M phase of the cell cycle, which may lead to opportunities to produce cloned pigs from piPSCs more efficiently.
authors.affiliation: Laboratory of Veterinary Embryology and Biotechnology, (VETEMBIO), Veterinary Medical Center and Collage of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea.|Institute for Stem Cell & Regenerative Medicine (ISCRM), Chungbuk National University, Cheongju, Chungbuk, Republic of Korea.
authors.forename: Eunhye
authors.initials: E
authors.lastname: Kim
authors.affiliation: Laboratory of Veterinary Embryology and Biotechnology, (VETEMBIO), Veterinary Medical Center and Collage of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea.
authors.forename: Zhong
authors.initials: Z
authors.lastname: Zheng
authors.affiliation: Laboratory of Veterinary Embryology and Biotechnology, (VETEMBIO), Veterinary Medical Center and Collage of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea.
authors.forename: Yubyeol
authors.initials: Y
authors.lastname: Jeon
authors.affiliation: Department of Animal Sciences, Agriculture, Life, & Environmental Sciences, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea.
authors.forename: Yong-Xun
authors.initials: YX
authors.lastname: Jin
authors.affiliation: Laboratory of Veterinary Embryology and Biotechnology, (VETEMBIO), Veterinary Medical Center and Collage of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea.|Institute for Stem Cell & Regenerative Medicine (ISCRM), Chungbuk National University, Cheongju, Chungbuk, Republic of Korea.
authors.forename: Seon-Ung
authors.initials: SU
authors.lastname: Hwang
authors.affiliation: Laboratory of Veterinary Embryology and Biotechnology, (VETEMBIO), Veterinary Medical Center and Collage of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea.
authors.forename: Lian
authors.initials: L
authors.lastname: Cai
authors.affiliation: Department of Agricultural Biotechnology, Research Institute for Agriculture and Life Science, Seoul National University, Seoul, Republic of Korea.
authors.forename: Chang-Kyu
authors.initials: CK
authors.lastname: Lee
authors.affiliation: Department of Animal Sciences, Agriculture, Life, & Environmental Sciences, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea.
authors.forename: Nam-Hyung
authors.initials: NH
authors.lastname: Kim
authors.affiliation: Laboratory of Veterinary Embryology and Biotechnology, (VETEMBIO), Veterinary Medical Center and Collage of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea.|Institute for Stem Cell & Regenerative Medicine (ISCRM), Chungbuk National University, Cheongju, Chungbuk, Republic of Korea.
authors.forename: Sang-Hwan
authors.initials: SH
authors.lastname: Hyun
mesh_terms: D000818:Animals; D001755:Blastocyst; D002478:Cells, Cultured; D019976:Cloning, Organism; D004171:Diploidy; D004622:Embryo, Mammalian / Q000166:cytology*; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D008677:Metaphase; D051379:Mice; D013552:Swine / Q000196:embryology*