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.
Highlights
Pigs are regarded as a powerful pre-clinical research tool because of their appropriate organ size, lifespan and similar anatomical and physiological attributes in comparison with humans, along with greater ease of use and availability compared to non-human primate models [1, 2]
Mouse Induced pluripotent stem cells (iPSCs) that were synchronized to metaphase by demecolcine treatment were used as donor cells to produce cloned mice and showed a better preimplantation developmental capacity than iPSCs at putative G1 stage and somatic cells [26, 27]
We speculated that by using pluripotent stem cells (piPSCs) synchronized to metaphase as donor cells, the preimplanation development capacity of piPSCs nuclear transfer (piPSNT) embryos could be improved, which may provide new possibilities to produce cloned pigs from piPSCs
Summary
Pigs are regarded as a powerful pre-clinical research tool because of their appropriate organ size, lifespan and similar anatomical and physiological attributes in comparison with humans, along with greater ease of use and availability compared to non-human primate models [1, 2]. Genetically modified pigs have many potential applications in agricultural and biomedical research [3]. The recent development of nucleases (CRISPR, TALENs and ZFN technologies) have enabled the highly efficient generation of knockout animals and a revolution in animal transgenesis [4,5,6]. Genetically modified pigs have been produced using genetically modified somatic cells and nuclear transfer (NT) [8]. Current somatic cell nuclear transfer (SCNT) efficiencies using pigs are relatively low, with only 1–3% developing to term without showing abnormalities at birth until now
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