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The aim of the present study was to determine whether porcine preadipocytes can be efficient donor cells for somatic cell nuclear transfer (SCNT) in pigs. Primary culture of porcine preadipocytes was established by de-differentiating mature fat cells taken from an adult pig. The cell cycle of the preadipocytes could be synchronized by serum starvation for 1 day, with a higher efficiency than control fetal fibroblasts. Incidence of premature chromosome condensation following nuclear transfer (NT) of preadipocytes was as high as that observed after NT with fetal fibroblasts. In vitro developmental rate of the NT embryos reconstructed with preadipocyte was equivalent to that of the fetal fibroblast derived embryos. Transfer of 732 NT embryos with preadipocytes to five recipients gave rise to five cloned piglets. These data demonstrate that preadipocyites collected from an adult pig are promising nuclear donor cells for pig cloning.

This paper methodologically compares the electro-fusion (EF) and intracytoplasmic injection (ICI) methods, as well as simultaneous fusion/activation (SA) and delayed activation (DA), in somatic nuclear transfer in pigs using fetal fibroblast cells. Comparison of the remodeling pattern of donor nuclei after nuclear transfer by ICI or EF showed that a high rate (80-100%) of premature chromosome condensation occurred in both cases whether or not Ca2+ was present in the fusion medium. Formation of pseudo-pronuclei tended to be lower for nuclear transfer performed by the ICI method (65% vs. 85-97%, p < 0.05). In vitro developmental potential of nuclear transfer embryos reconstructed with IVM oocytes using the EF method was higher than that of those produced by the ICI method (blastocyst formation: 19 vs. 5%, p < 0.05), and it was not improved using in vivo-matured oocytes as recipient cytoplasts. Embryos produced using SA protocol developed to blastocysts with the same degree of efficiency as those produced under the DA protocol (11 vs. 12%). Use of the EF method in conjunction with SA was shown to be an efficient method for producing cloned pigs based on producing a cloned normal pig fetus. However, subtle differences in nuclear remodeling patterns between the SA and DA protocols may imply variations in their nuclear reprogramming efficiency.

Abstract. Four methods of cell cycle synchronization of porcine preadipocytes for use as nuclear donors in somatic cell cloning were compared: serum starvation, differentiation induction, contact inhibition and roscovitine treatment. After three days of differentiation induction, the percentage of nuclear donor cells synchronized at the G0/G1 phase reached a peak value of 91.8%, which was significantly higher (P<0.05) than the percentage attained by serum starvation (84.9-89.8%), contact inhibition (78.3-83.7%) or roscovitine treatment (67.8-80.3%). Cell cycle synchronization by serum starvation, contact inhibition and roscovitine treatment all increased the percentage of apoptotic cells, while no increase was observed when the donor-cell cycle was synchronized by differentiation induction (Annexin V-positive: 15.7% to 19.3% vs. 7.7%, P<0.05; TUNEL-positive: 12.8% to 14.0% vs. 8.3%, P<0.05). Additionally, comparison of the in vitro development of nuclear transfer (NT) embryos formed from the nuclei of differentiation-induced or serum-starved preadipocytes revealed that, in both cases, a high proportion of embryos developed to the blastocyst stage (39.0 and 33.7%, respectively). In this study, NT embryos reconstructed with preadipocytes synchronized by differentiation induction were transferred to four recipient pigs, three of which gave birth to a total of 17 piglets (4.2%, 17/403). These results demonstrate that donor-cell cycle synchronization by differentiation induction enables effective production of cloned pigs. The findings also indicate that differentiation induction of multipotent cells is an excellent method of cell cycle synchronization that permits highly efficient synchronization of cells at the G0/G1 phase. Key words: Cell cycle synchronization, Multipotent cell, Nuclear transfer, Pig, Preadipocyte, Somatic cell cloning (J. Reprod. Dev. 55: [121][122][123][124][125][126][127] 2009) igs are not only raised as livestock for human consumption, but are also gaining recognition as valuable experimental animals. Expectations are particularly high for the use of cloned and genetically modified pigs produced by somatic cell nuclear transfer (SCNT) in cutting-edge medical research. Already, α1,3-galactosyltransferase gene knockout pigs have been produced as organ donors for xenotransplantation [1][2][3][4][5][6][7][8]. In addition, transgenic pigs serving as human disease models, such as those carrying the gene for endothelial cell nitric oxide synthase (eNOS), have been developed [9]. To further advance the use of cloned and genetically modified pigs in research, however, a SCNT system that enables more efficient generation of embryos is needed.In SCNT, the type and cell cycle phase of the donor cell are important factors that affect the efficiency of generating cloned animals [10][11][12][13][14]. In previous work, we demonstrated that preadipocytes have characteristics that render them excellent nuclear donor cells in porcine SCNT [15]. We have successfully produced cloned piglets by nuclear transfer ...

Abstract. Porcine nuclear transfer embryos were reconstructed by intracytoplasmic nuclear injection of cumulus cell nuclei into recipient oocytes matured in vitro by two types of method (NCSU23-based and TCM199-based methods). Remodelling of the transferred nuclei and in vitro development of the reconstructed embryos were investigated. Intracytoplasmic nuclear injection did not induce activation of the recipient oocytes regardless of the type of maturation method (NCSU23-based: 61/ 61, 100%, TCM199-based: 49/50, 98%). When the reconstructed embryos were examined 1 h after nuclear injection many of them (NCSU23-based: 22/29, 76%; TCM199-based: 19/23, 83%) possessed condensed chromosomes or a chromosome array resembling the maternal metaphase plate. The proportion of the reconstructed embryos possessing condensed chromosomes arranged in a scattered fashion showed a tendency to increase, when they were examined 2-5 h after nuclear injection. Reconstructed embryos were electrically activated either 2-2.5 h or 3.5-4 h after nuclear injection, followed by fixation/staining to examine formation of pronucleus-like nuclei (pseudo-nuclei). Regardless of the nuclear injection/activation interval tested, 68% (36/53) of the reconstructed embryos developed pseudo-pronuclei ranging in number between 1 and 4. When the reconstructed embryos were cultured for 7 days to assess their developmental competence, 5 (5/98) to 11% (12/111) developed to blastocysts regardless of the type of the recipient oocyte and nuclear injection/activation intervals. These results show that intracytoplasmic injection of porcine cumulus cell nuclei achieves a high rate of nuclear remodelling and that the reconstructed embryos are capable of developing into blastocysts.