An Improved System for Generation of Diploid Cloned Porcine Embryos Using Induced Pluripotent Stem Cells Synchronized to Metaphase

PLoS One. 2016 Jul 29;11(7):e0160289. doi: 10.1371/journal.pone.0160289. eCollection 2016.

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.

MeSH terms

  • Animals
  • Blastocyst
  • Cells, Cultured
  • Cloning, Organism*
  • Diploidy*
  • Embryo, Mammalian / cytology*
  • Induced Pluripotent Stem Cells / cytology*
  • Metaphase*
  • Mice
  • Swine / embryology*

Grants and funding

This work was supported, in part, by a grant from the “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ011288, PJ011077)" Rural Development Administration, the ”Ministry of Trade, Industry & Energy(MOTIE), Korea Institute for Advancement of Technology(KIAT) through the Leading Industry Development for Economic Region (Project No. R0004357)“ and “Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Advanced Production Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (Grant number: 115103-02)”, Republic of Korea. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.