• Asian-Australasian Journal of Animal Sciences
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• v.21 no.11
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• pp.1665-1672
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• 2008

Interspecies somatic cell nuclear transfer (iSCNT) is a useful method to preserve endangered species and to study the reprogramming event of a nuclear donor cell by the oocyte. Although several studies of iSCNT using murine cells and bovine oocytes have been reported, the development of murine-bovine iSCNT embryos beyond the 8-cell stage has not been successful. In this paper, we examined the developmental potential of embryos reconstructed with a murine embryonic fibroblast as the nuclear donor and a bovine oocyte as the cytoplasm recipient. The reconstructed embryos were cultured in CZB (murine medium) or CR1aa (bovine medium). In addition, for the development of a murine-bovine iSCNT blastocyst, the antioxidant ${\beta}$-mercaptoethanol (${\beta}ME$) was supplemented to CR1aa medium. Furthermore, to verify the mouse genome activation in murine-bovine iSCNT embryos, RT-PCR analysis of murine Xist was performed. The development of the murine-bovine iSCNT embryos cultured in CR1aa was significantly higher than that in CZB (p<0.05). With respect to the effect of BME on the development of the murine-bovine iSCNT blastocyst, addition of BME produced a significant increase in blastocyst development (p<0.05). Karyotype analysis confirmed that the reconstructed embryos were derived from murine cells (40XX). The Xist gene was gradually increased from the 8-cell stage to the blastocyst stage. This is the first report of blastocyst development of iSCNT embryos derived from murine somatic cells and bovine oocytes. These results demonstrate that bovine cytoplasm can support the development of later stages of a preimplantation embryo from murine-bovine iSCNT.

• Journal of Embryo Transfer
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• v.30 no.4
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• pp.345-350
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• 2015

In the last 10 years, porcine somatic cell nuclear transfer to generate transgenic pig has been performed tremendous development with introduction and knockout of many genes. However, efficiency of porcine somatic cell nuclear transfer is still low and embryo transfer (ET) is one of important step for production efficiency. In porcine ET for production of transgenic cloned pig, we can consider many of points to increase production rates. In respect of seasonality and weather, porcine ET usually is not performed in summer and winter. Cloned transgenic embryos must be transferred into reproductive tracts of recipients where embryos are located after natural fertilization with similar estrous cycle. If cloned embryos with 2~4 cell stage are transferred, they must be transferred into oviducts in periovulatory stage. Number and deposition sites of transferred cloned embryos are important. And we must compare the methods of ET between surgical and non-surgical ones in respect of production efficiency. Sow recipients after natural estrus is most preferred recipients however its cost is must be considered. Here we will review many of current studies about porcine embryo transfer to increase production efficiency of transgenic pigs and strategies for further studies.

• Journal of Embryo Transfer
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• v.31 no.2
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• pp.131-136
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• 2016

Since the first success of animal cloning, somatic cell nuclear transfer presented various ideas in many research areas such as regenerative medicine. However, SCNT embryos has poor survival rate. Therefore, numerous researches carried out to enhance the developmental capability of porcine nuclear transfer embryos. Cytochalasin B, demecolcine, latrunculin A, cycloheximide and 6-dimethylaminopurine are efficient chemicals treated in post-activation procedure to increase the efficiency of SCNT. This review study is aim to investigate the effects of these chemicals applied to post-activation in porcine SCNT. Cytochalasin B, demecolcine, latrunculin A are cytoskeletal manuplators inhibit extrusion of pseudo-polar body. Cytochalasin B and demecolcine showed considerably higher blastocyst formation proportion (26-28%) compared to when they are not treated (16%). And when latrunculin A was treated for postactivation, blastocyst formation proportion was increased in SCNT embryos exposed to LA (38%) than those in control (14%). On the other hand, cycloheximide and 6-dimethylaminopurine are protein synthesis and kinase inhibitors. And they help to maintain $Ca^{2+}$ fluctuation in oocytes. Cleavage and blastocyst rates of NT embryos were increased when they were exposed to CHX (16.9% and 5.4% with no CHX).And 6-DMAP also showed higher blastocyst formation (21.5% compared to 15.7%, control). Although all these chemicals have different mechanisms, they showed developmental competence enhancement in NT embryos. However, there are only few studies comparing each chemical's post-activation effect. Therefore, further research and study should be conducted to find optimal chemical for improving the efficiency of SCNT.

• Reproductive and Developmental Biology
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• v.36 no.1
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• pp.49-53
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• 2012

The present study was conducted to examine the generation of reactive oxygen species (ROS) during micromanipulation procedures in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine enucleated oocytes were electrofused with donor cells, activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. Oocytes and embryos were stained in dichlorodihydrofluorescein diacetate or 3'-(p-hydroxyphenyl) fluorescein dye and the $H_2O_2$ or $^.OH$ radical levels were measured. $In$ $vitro$ fertilization (IVF) was performed for controls. The samples were examined with a fluorescent microscope, and fluorescence intensity was analyzed in each oocyte and embryo. The $H_2O_2$ and $^.OH$ radical levels of reconstituted oocytes were increased during manipulation (37.2~49.7 and 51.0~55.2 pixels, respectively) as compared to those of mature oocytes ($p$<0.05). During early $In$ $vitro$ culture, the ROS levels of SCNT embryos were significantly higher than those of IVF embryos ($p$<0.05). These results suggest that the cellular stress during micromanipulation procedures can generate the ROS in bovine SCNT embryos.

• Journal of Embryo Transfer
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• v.15 no.1
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• pp.23-31
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• 2000

This study was conducted to investigate the effects of quiescent treatment of donor cells and activation treatment time of recipient cytoplasm on nuclear remodeling and in vitro development of somatic cell-cloned bovine embryos. Serum starved, confluent and nonquiescent cycling adult skin cells were teansferred into enucleated oocytes. Nuclear transfer oocytes were activated at 30 min, 1 and 2 hrs after electrofusion. Some nuclear transfer embryos(23% to 35%) extruded a polar body, which was not affected by quiescent treatment of donor cells and activiation time of recipient cytoplasm. About 68% of nuclear transfer embryos fused with a serum starved cells has a chromatin clump, but which was not different from embryos fused with confluent(51%) and nonquiescent(47%) cells. The proportion of embryos with a single chromatin clump was sightly increased when nuclear transfer embryos were activated within 30 min after fusion(69%) compared to those were activated at 1 and 2 hrs after fusion, but there was not significantly different. Development rates to the blastocyst stage were 8.6% and 15.9% when serum starved and confluent cells were transferred, which were higher than that of control group. Developmental rate to the blastocyst stage was higher in embryos were activated within 30 min after fusion (17.3%) compared to those of embryos were activated at 1 and 2 hrs after fusion (P<0.05). From the present result, it is suggested that quiescent treatment of donor cells and activation time of recipient cytoplasm can affect the in vitro development. Quiescent plasm activation within 30 min after fusion could increase the number of embryos with a normal chromation structure, which results in increased in vitro development.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.105-113
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• 2011

The present study investigated the nuclear remodeling, development potential with telomerase activity and transcription level of X-linked genes (ANT3, HPRT, MeCP2, RPS4X, XIAP, XIST and ZFX) in the bovine somatic cell nuclear transfer (SCNT) embryos using two different fusion and activation methods. Female adult fibroblasts were injected into perivitelline space of in vitro matured oocytes. The oocyte-nucleus complexes were fused and followed by immediately either activated (Group 1), or activated at 1 h post-fusion (hpf) (Group 2), respectively. The incidence of normal premature chromosome condensation (PCC) at 1 hpf was slightly increased in the Group 2, compared to those of Group 1, but there was no significant (p<0.05) difference. The incidence of normal pronucleus (PN) and chromosome spread at 5 and 18 hpf were significantly (p<0.05) higher in the Group 2 than those of Group 1. The cleavage rate to 2-cell stage, developmental rate to blastocyst stage, and the mean number of total and ICM cell numbers were significantly (p<0.05) higher in the Group 2, compared to those of Group 1. Level of telomerase activity was significantly (p<0.05) higher in the SCNT blastocysts of Group 2, compared to those of Group 1. Transcript levels of HPRT, MeCP2 and XIST were not significantly (p<0.05) different between blastocysts of Group 1 and 2. However, transcript level of ANT3, RPS4X, XIAP and ZFX were significantly (p<0.05) up-regulated in the SCNT blastocysts of Group 2, compared to those of Group 1. Taken together, it is concluded that oocyte activation at 1 hpf induces the enhanced developmental potential by efficient nuclear remodeling and subsequent facilitation of the nuclear reprogramming of bovine SCNT embryos.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2005.10a
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• pp.86-86
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• 2005

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2005.10a
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• pp.87-88
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• 2005

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2005.10a
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• pp.89-90
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• 2005

• Journal of Embryo Transfer
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• v.30 no.4
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• pp.305-313
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• 2015

Xenotransplantation of pig islet regarded as a good alternative to allotransplantation. However, cellular death mediated by hypoxia-reoxygenation injury after transplantation disturb success of this technique. In the present study, we produce transgenic pig expressing human heme oxygenase 1 (HO1) genes to overcome cellular death for improving efficiency of islet xenotransplantation. Particularly, Korean miniature pig breed, Micro-Pig, was used in the present study. Somatic cell nuclear transfer (SCNT) technique was used to produce the HO1 transgenic pig. Six alive transgenic piglets were produced and all the transgenic pigs were founded to have transgene in their genomic DNA and the gene was expressed in all tested organs. Also, in vitro cultured fibroblasts derived from the HO1 transgenic pig showed low reactive oxygen species level, improved cell viability and reduced apoptosis level.