Pseudophysiological transcomplementary activation of reconstructed oocytes as a highly efficient method used for producing nuclear-transferred pig embryos originating from transgenic foetal fibroblast cells

Abstract

The completely new strategy of pseudophysiological transcomplementary (transcytoplasmic) activation (PP-TCA) of nuclear-transferred oocytes, which had been derived from pWAPhGH-GFPBsd transfected foetal fibroblast cells, was recently applied to the somatic cell cloning of pigs. It resulted in the considerable enhancing not only the cleavage activity of cultured cloned embryos, but also their morula and blastocyst formation rates as compared to the use of standard simultaneous fusion and electrical activation of reconstituted oocytes (77% vs. 57%, 63% vs. 46% and 40% vs. 27%, respectively). Altogether, the use of cytosolic components descended from heterologous (rabbit) zygotes as the agents for stimulation of porcine clonal cytoplasmic hybrids (cybrids) turned out to be reliable and feasible strategy for the generation of transgenic blastocysts by somatic cell nuclear transfer (SCNT). Furthermore, to our knowledge, no previous study has reported the preimplantation developmental outcome of transgenic nuclear-transferred pig embryos following the PP-TCA that was developed and optimised in our laboratory. 2003a, Brunetti et al. 2008, whose meiotic cell cycle is reversibly blocked at the second metaphase (MII) stage. One of the most important factors that significantly affect the developmental competences of porcine cloned embryos is the artificial activation of oocytes reconstructed with somatic cell nuclei. The ability of an artificial stimulus to

Successful cloning of an adult breeding boar from the novel Chinese Guike No. 1 swine specialized strain

“…Investigations have been addressed that the developmental competences of porcine SCNT-derived embryos are affected by a large number of factors. Among them, an important role seems to be played by the quality of in vitro-matured pig oocytes used as nuclear recipient cells (Dang-Nguyen et al 2011 ; Ju and Rui 2012 ; Pribenszky et al 2012 ; Alvarez et al 2015 ), different approaches to SCNT procedures (Samiec et al 2003 , 2012 ; Li et al 2004 ; Nánássy et al 2008 ; Samiec and Skrzyszowska 2010b , 2012b , 2014a ) as well as the systems applied to in vitro embryo culture (Nánássy et al 2008 ; Yoshioka et al 2008 ; Yoshioka 2011 ). In addition, evidences also suggest the nuclear donor cell type plays a pivotal role in pig cloning (Hao et al 2009 ; Lee et al 2010 ; Samiec and Skrzyszowska 2010a ; Richter et al 2012 ; Fan et al 2013 ; Wei et al 2013 ; Liu et al 2014 , 2015 ; Samiec et al 2015 ).…”

Trichostatin A-Mediated Epigenetic Transformation of Adult Bone Marrow-Derived Mesenchymal Stem Cells Biases theIn VitroDevelopmental Capability, Quality, and Pluripotency Extent of Porcine Cloned Embryos

“…The approaches to somatic cell cloning and in vitro culture of nuclear-transferred pig embryos that were used in the current experiments were thoroughly presented in our previous studies [ 15 , 16 , 18 , 59 , 60 ]. Concisely, prior to the SCNT procedure, cumulus-denuded in vitro matured gilt/sow oocytes were incubated in the maturation medium supplemented with 0.4 μ g/mL demecolcine (DMCC) for 50 to 60 min.…”

Chlorogenic acid supplementation during in vitro maturation improves maturation, fertilization and developmental competence of porcine oocytes

“…Currently used IVP systems comprise three major steps: in vitro maturation (IVM), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) and in vitro culture (IVC) of fertilized or cloned embryos (Samiec, Opiela, Lipiński, & Romanek, ; Samiec & Skrzyszowska, , ; Somfai & Hirao, ). Although many attempts have been made to produce high‐quality IVF‐ or SCNT‐derived embryos, their developmental competence in the pig remains insufficient and lower than that of in vivo‐derived embryos of the pig and of other mammalian species, such as cattle or mice (Dang‐Nguyen et al., ; Kikuchi et al., , ; Samiec, Skrzyszowska, & Lipiński, ). Thus, it is crucial to improve the developmental competence of IVF‐ or SCNT‐derived embryos because this inefficiency diminishes its application to further studies that require high‐quality embryos (Pang et al., ; Samiec & Skrzyszowska, , ; Yoshioka, Suzuki, Tanaka, Anas, & Iwamura, ).…”