The present study was carried out to determine (1) the optimal duty cycle of ultrasound for activation of pig oocytes and cloned embryos derived from miniature pig fetal fibroblasts and (2) whether cloned embryos can develop to term following activation by ultrasound stimulation. When oocytes were exposed to ultrasound with 20% or 30% duty cycle, the blastocyst formation rates were significantly (P < 0.05) higher than that of oocytes exposed to ultrasound with 10% duty cycle. In contrast, the blastocyst formation rate of cloned embryos decreased as the duty cycle of ultrasound increased; the value of embryos exposed to ultrasound with 10% duty cycle was significantly (P < 0.05) higher than that of embryos exposed to ultrasound with 50% duty cycle. When cloned embryos exposed to ultrasound with 10% duty cycle were transferred into the oviducts of two recipient gilts to assess their development in vivo, the pregnancy of one of the gilts was maintained to term and two piglets were delivered via Cesarean section. The results of the present study showed that (1) although the duty cycle of ultrasound affects in vitro development after activation of both pig oocytes and miniature pig cloned embryos, the optimal duty cycle is different between them and (2) miniature pig cloned embryos have the ability to develop into piglets after activation by ultrasound stimulation.

This IB4-SAP-mediated method of selection of alphagal epitope-negative cells will provide an alternative to the present method of cytotoxicity-based selection using specific antibody and complement.

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The present study was carried out to examine the effects of post-activation treatment of trichostatin A (TSA), a histone deacetylase inhibitor, on in vitro development and transgene function of somatic cell nuclear transfer (SCNT) embryos derived from Clawn miniature pig embryonic fibroblast (PEF) transfected with a bacterial endo-b-galactosidase C gene (removal of the a-galactosyl (Gal) epitope). SCNT embryos were incubated with or without TSA (50 or 100 nmol/L) after activation, cultured in vitro and assessed for cleavage, blastocyst formation and transgene function. The rate of blastocyst formation was significantly higher in SCNT embryos treated with 50 nmol/L TSA than that in control (P < 0.05), whereas the rate of cleavage and cell number of blastocyst did not differ. Following labelling with fluorescein isothiocyanate-labelled BS-I-B4 isolectin, the intensity of fluorescence observed on cell-surface was dramatically reduced in transgenic SCNT blastocyst in comparison with non-transgenic SCNT blastocyst. However, the reduction of a-Gal epitope expression in transgenic SCNT blastocyst was not affected by TSA treatment. The results of this study showed that post-activation treatment with 50 nmol/L TSA is effective to improve in vitro developmental capacity of transgenic SCNT miniature pig embryos without the modification of transgene function.

Abstract. The objective of this study was to examine the effect of postactivation treatment with latrunculin A (LatA), an actin polymerization inhibitor, on in vitro and in vivo development of somatic cell nuclear transfer (SCNT) embryos derived from kidney fibroblasts of an aged Clawn miniature boar (12 years old). After electric activation, SCNT embryos were treated with 0, 0.5 or 1 μM LatA and cultured in vitro. The rate of blastocyst formation was significantly higher (P<0.05) in SCNT embryos treated with 0.5 μM LatA (38%) than those in control (14%). When cloned embryos treated with 0.5 μM LatA were transferred into the oviducts of two recipient miniature gilts to assess their development in vivo, both recipients became pregnant; one maintained pregnancy to term, and a live piglet (weighing 220 g) was delivered by Caesarean section. The results of this study indicated that the postactivation treatment with LatA was effective in improving in vitro developmental capacity of SCNT miniature pig embryos derived from kidney fibroblasts of an aged animal and that miniature pig cloned embryos treated with LatA had the ability to develop to term. Key words: Aged donor, Clawn miniature pig, Cytoskeletal inhibitors, Latrunculin A, Nuclear transfer (J. Reprod. Dev. 58: [398][399][400][401][402][403] 2012) C lawn miniature pigs (inbred strain) have a potential value for biomedical research and xenotransplantation due to their clear genetic background [1][2][3] and few individual differences; thus, they have stimulated investigation of novel strategies directed at generating a genetically modified miniature pig by somatic cell nuclear transfer (SCNT). We have shown how to produce live offspring derived from Clawn miniature pig SCNT embryos; however, the efficiency still remains low [4]. To date, pigs have been cloned with SCNT using donor cells derived from fetal pigs to pigs several years of age. The efficiency of SCNT is influenced by many factors, including quality of recipient oocytes, type of donor cells, reprogramming of donor nuclei and condition of recipient gilts [4][5][6][7][8][9][10][11][12][13]. In particular, the diploid complement retention is one of the most important factors in improving the developmental ability of SCNT embryos [14,15]. SCNT embryos derived from diploid cells are generally treated with cytoskeletal inhibitors, such as cytochalasin B (CB), after activation to prevent extrusion of a pseudo polar body (pPB) and the possible loss of chromosomes maintain normal ploidy [7,16,17]. Several reports have shown that CB treatment of SCNT embryos resulted in increased blastocyst formation in vitro [18][19][20][21]. However, it has been reported that postactivation treatment with CB had no effect on either blastocyst rate or cell number of SCNT embryos [9,22]. CB possess cytotoxicity [23], and its cytotoxicity might have a detrimental effect on the subsequent development of SCNT embryos.Latrunculin A (LatA), a toxin purified from the Red Sea sponge Latrunculia magnifica, is a member of the cytoskel...

This study was carried out to examine the effect of postactivation treatment with latrunculin A (LatA), an actin polymerisation inhibitor, on in vitro and in vivo development of somatic cell nuclear transfer (SCNT) embryos derived from gene-modified Clawn miniature pig cells. After the fusion and activation, SCNT embryos were treated with or without a cytoskeletal inhibitor [LatA or 10.4 microM cytochalasin B (CB) for 2 h]. The cleavage rate was significantly higher (p < 0.05) in embryos exposed to 0.5 microM LatA than those in embryos exposed to CB and without a cytoskeletal inhibitor. Moreover, the blastocyst formation rate was significantly higher (p < 0.05) in embryos exposed to 0.5 or 1 microM LatA than those in embryos exposed to CB and without a cytoskeletal inhibitor. In addition, five fetuses were obtained from recipient uteri after transfer of embryos treated with 0.5 muM LatA. The results of this study show for the first time that postactivation treatment with LatA is effective to improve in vitro developmental capacity of gene-modified cloned miniature pig embryos and embryos treated with LatA have the ability to develop into fetuses.

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Abstract. Reversine, a 2-(4-morpholinoanilino)-6-cyclohexylaminopurine analog, can induce dedifferentiation of myogenic lineage-committed cells into multipotent mesenchymal progenitor cells, from which osteoblasts and adipocytes redifferentiate under lineage-specific inducing conditions. Although the molecular mechanism of how reversine causes dedifferentiation of a differentiated cell has not been fully elucidated, we speculated that it would be involved in reprogramming. In the present study, we examined whether reversine can enhance the development of somatic cell nuclear transfer (SCNT) embryos by improving the reprogramming state of the somatic cell nuclei. As donor cells, we used miniature pig fetal fibroblasts transfected with a plasmid construct containing a mouse Oct-3/4 promoter and enhanced green fluorescent protein (EGFP) cDNA. When the nuclei of these transfected cells are reprogrammed to an undifferentiated state in the SCNT embryos, EGFP expression is expected to commence under the control of the Oct-3/4 promoter. After SCNT, the resulting embryos were treated with 5 μM reversine for different durations (0, 6, 12, 18 and 24 h) or at different concentrations (0, 1, 5 and 10 μM) of reversine for 12 h and then cultured in vitro. When embryos were treated with 5 μM reversine for 12 h, the blastocyst formation rate was significantly (P<0.01) higher than that of embryos without reversine treatment. However, the strength and pattern of EGFP expression in the embryos were not affected by the same treatment. A normal-looking fetus was obtained 21 days after transfer of embryos treated with 5 μM reversine for 12 h into recipients. The present findings indicate that treatment with reversine is beneficial for enhancement of the in vitro development of miniature pig SCNT embryos, although the underlying mechanism is still unclear. Key words: In vitro development, Miniature pig, Reprogramming, Reversine, Somatic cell nuclear transfer (J. Reprod. Dev. 56: [291][292][293][294][295][296] 2010) ecently, Chen et al. [1] reported that a 2-(4-morpholinoanilino)-6-cyclohexylaminopurine analog could induce dedifferentiation of myogenic lineage-committed C2C12 cells to become multipotent mesenchymal progenitor cells in the concentration range of 1-10 μM. These progenitor cells could redifferentiate into osteoblasts or adipocytes under appropriate differentiation inducing conditions. The compound was named "reversine" because of its ability to reverse terminally differentiated cells to progenitor cells [1]. More recently, it was reported that human fibroblasts treated with reversine exhibited redifferentiation into skeletal muscle cells in vitro and in vivo [2]. Thus, reversine has the ability to reprogram somatic cells to a state of diverse plasticity that can be manipulated to differentiation into different cell types.Low cloning efficiency, which is often associated with low developmental rate of somatic cell nuclear transfer (SCNT) embryos, remains the major obstacle to use this technology in various fields of ani...