• Proceedings of the KSAR Conference
• /
• 2004.06a
• /
• pp.218-218
• /
• 2004

This study was designed to investigate the in vitro developmental ability and apoptosis of embryos nuclear transferred (NT) with frozen-thawed (FT) or cooled donor cells in bovine. Cultured adult bovine ear cells were used as donor cells at confluent condition (CC), after cooling at 4℃ for 48 hour, or after FT. (omitted)

• Korean Journal of Animal Reproduction
• /
• v.27 no.4
• /
• pp.349-357
• /
• 2003

Although nuclear transfer (NT) techniques are used to clone animals, its efficiency is very low. Moreover, nuclear transfer has resulted in offspring with severe developmental problems, probably due to incomplete nuclear reprogramming. Nuclear reprogramming is characterized by functional modification of the transferred nucleus to allow it to direct normal embryo development with the potential to grow to term. Although the nature of the reprogramming factor(s) in mammals is not clear, various nuclear as well as cytoplasmic components are involved in the processes. In this article we review recent data on factors involved in the nuclear reprogramming of cloned embryos.

• Proceedings of the Korean Society of Embryo Transfer Conference
• /
• 2006.10a
• /
• pp.54-55
• /
• 2006

• Reproductive and Developmental Biology
• /
• v.35 no.1
• /
• pp.67-75
• /
• 2011

The faulty regulation of imprinting gene lead to the abnormal development of reconstructed embryo after nuclear transfer. However, the correlation between the imprinting status of donor cell and preimplantation stage of embryo development is not yet clear. In this study, to determine this correlation, we used the porcine spermatogonial stem cell (pSSC) and fetal fibroblast (pFF) as donor cells. As the results, the isolated cells with laminin matrix selection strongly expressed the GFR ${\alpha}$-1 and PLZF genes of SSCs specific markers. The pSSCs were maintained to 12 passages and positive for the pluripotent marker including OCT4, SSEA1 and NANOG. The methylation analysis of H19 DMR of pSSCs revealed that the zinc finger protein binding sites CTCF3 of H19 DMRs displayed an androgenic imprinting pattern (92.7%). Also, to investigate the reprogramming potential of pSSCs as donor cell, we compared the development rate and methylation status of H19 gene between the reconstructed embryos from pFF and pSSC. This result showed no significant differences of the development rate between the pFFs ($11.2{\pm}0.8%$) and SSCs ($13.3{\pm}1.1%$). However, interestingly, while the CTCF3 methylation status of pFF-NT blastocyst was decreased (36.3%), and the CTCF3 methylation status of pSSC-NT blastocyst was maintained. Therefore, this result suggested that the genomic imprinting status of pSSCs is more effective than that of normal somatic cells for the normal development because the maintenance of imprinting pattern is very important in early embryo stage.

• Reproductive and Developmental Biology
• /
• v.29 no.2
• /
• pp.83-91
• /
• 2005

Somatic cell nuclear transfer in cattle has limited efficiency in terms of production of live offspring due to high incidence of fetal failure after embryo transfer to recipients. Such low efficiency of cloning could possibly arise from abnormal and poorly developed placenta. In the present study the placental proteome in late pregnancy established from in vitro fertilization (IVF) and nuclear transfer (NT) was analysed. Proteome alternation was tested using two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- TOF). Comparing placenta from NT embryos to those from IVF counterparts, significant changes in expression level were found in 18 proteins. Of these proteins 12 were not expressed in NT placenta but expressed in IVF counterpart, whereas the expression of the other 6 proteins was limited only in NT placenta. Among these proteins, cytokeratin 8 and vimentin are considered to be involved in regulation of post-implantation development. In particular, cytokeratin 8 and vimentin may be used as makers for placental development during pregnancy because their expression levels changed considerably in NT placental tissue compared with its IVF counterpart. Data from 2-DE suggest that protein expression was disorientated in late pregnancy from NT, but this distortion was eliminated with progression of pregnancy. These findings demonstrate abnormal placental development during late pregnancy from NT and suggest that alterations of specific placental protein expression may be involved in abnormal function of placenta.

• Korean Journal of Animal Reproduction
• /
• v.26 no.2
• /
• pp.173-182
• /
• 2002

The mechanisms underlying of the visual assessment and resulting in optimum embryonic development following in vitro maturation, fertilization, and culture are unclear, It was known that in vitro produced embryos show more frequent occurrence of fragmentation, which resulted in poor developmental potential and decreased implantation rate. The objective of this study was to investigate the apoptotic rates in bovine blastocyst derived from in vitro fertilization (IVF) and nuclear transfer (NT). In addition, the expression levels of Bcl-2 and Bax gene were investigated in the blastocyst to confirm their potential roles in the regulation of apoptosis during preimplantation embryonic development. Analysis of apoptosis was carried out by using terminal deoxynucleotidyl transferase mediate dUTP nick end labeling (TUNEL) method. The levels of Bcl-2 and Bax gene in the blastocyst derived from IVF and NT were determined by RT-PCR. The proportion of TUNEL positive signal in blastocyst derived from NT was significantly higher than that in blastocyst derived from IVF (p<0.001). Bcl-2 expression level of blastocyst derived from IVF was higher than that of blstocyst derived from NT. However, high expression level of Bax was observed in the blastocyst derived from NT. These results indicates that apoptosis is more responsible for fiagmentation in bovine blastocyst derived from NT than IVF. These results suggested that the increase of developmental failure followed by NT could be caused by nuclear fragmentation as apoptosis.

• Korean Journal of Animal Reproduction
• /
• v.20 no.1
• /
• pp.9-17
• /
• 1996

This study was conducted to investigate freezability of in vitro and in vitro matured rabbit oocytes, possibility of NT using frozen-thawed unfertilized oocytes, and NT efficiency by zona-slit micromanipulation. After freezing of in vitro matured oocytes, 33 to 49% of oocytes appeared normal morphology and 1.0M DMSO and 1.5M glycerol showed slightly high survival rate, but there was no difference in survival between two cryoprotectants. Freezability of in vitro matured oocytes was low in 1.5M glycerol and more sensitive to freezing. Efficiency of enucleation and fusion rate in method B was higher than that in method A and no difference in this efficiency was between 3 groups of oocytes in method B. Cleavage rate and developmental capacity to M＋B stage of fused embryos derived from frozen oocytes was greatly lower than that from fresh oocytes, respectively(39.1% : 79.5% ; 3.1% : 19.3%) and there was no difference in cleavage rate between DC voltages in two group oocytes. Additional incubation in cytochalasin B after electrical stimulation did not affect embryo development. In conclusion, it is suggested that enucleation and nucelar transfer by slitting of zona is more effective method in rabbit and that further study on optimum freezing conditions for in vitro matured oocytes is necessary to use as recipient oocytes.

• Asian-Australasian Journal of Animal Sciences
• /
• v.20 no.4
• /
• pp.487-495
• /
• 2007

The objectives of the present study were to initiate cloning of Korean native goat by somatic cell nuclear transfer (NT) and to examine whether unovulated (follicular) oocytes can support the same developmental ability of NT embryos as ovulated (oviductal) oocytes after hCG injection in stimulated cycles of the goat. The in vivo-matured and immature oocytes were collected from the oviducts and follicles of superovulated does, respectively, and the immature oocytes were maturated in vitro. Ear skin fibroblasts derived from a 3-yr-old female Korean native goat were used as the donors of nuclei or karyoplasts. Following fusion, activation and in vitro culture to a 2- to 4-cell stage, 49 in vitro-derived and 105 in vivo-derived embryos were transferred to 6 and 17 recipient does, respectively. One doe and three does of the respective groups were identified as pregnant by ultrasonography on day 30 after embryo transfer. However, only one doe, which had received in vivo-derived embryos, delivered a normal female kid of 1.9 kg on d 149. The cloned kid gained more weight than her age-matched females as much as 87% during the first 4 mo after birth (17.7 vs. $9.4{\pm}0.8$ kg) and reached puberty at 6-mo age a few months earlier than normal female does. The telomere length of the kid, which was similar to that of the donor fibroblast at 2-mo age, decreased 8% between 2- and 7-mo ages. Moreover, at 7-mo age, she had 21% shorter telomere than her age-matched goats. To our knowledge, this is the first case in which a cloned animal born with a normal weight exhibited accelerated growth and development. The unusually rapid growth and development of the cloned goat may have resulted from SCNT-associated epigenetic reprogramming involving telomere shortening.

• Korean Journal of Animal Reproduction
• /
• v.27 no.1
• /
• pp.25-34
• /
• 2003

Human thrombopoietin (hTPO) is a cytokine that plays a central role in megakaryopoiesis. To direct hTPO expression in the mammary gland, an expression vector was constructed by combining the promoter of bovine beta-casein gene, cDNA of hTPO and neomycin resistance gene (pBT-L neo). Fibroblast cells derived from cow's ear skin tissue were transfected with the expression vector (pBT-L neo) using Lipofectamine. Transfected cells resistant to G418 trea？nt were cultured to form the colonies for more than 2 weeks. The transformed colonies identified by PCR were further expanded prior to nuclear transfer. Reconstructed oocytes with transformed cells were electrofused, activated using calcium ionophore and 6-DMAP, and cultured in vitro for 7 days. Of 35 cell colonies analyzed by PCR, 29 colonies (82.9%) were positive for the hTPO gene. Cleavage and developmental rates to the blastocyst stage of reconstructed embryos with the transformed cells were 65.1% and 23.8%, respectively Of 29 blastocysts that developed from reconstructed embryos with the transformed cells, 27 embryos (93.1%) were transgenic. These results indicate that transgenic bovine embryos can be efficiently produced by somatic cell nuclear transfer using transformed cells.

• Proceedings of the KSAR Conference
• /
• 2002.06a
• /
• pp.39-39
• /
• 2002

The mechanisms underlying the visual assessment and resulting in optimum embryonic development following in vitro maturation, fertilization, and culture are unclear. It is known that in vitro produced embryos show more frequent occurrence of fragmentation, which result in poor developmental potential and decreased implantation rate. The objective of this study was to investigate the apoptotic rates in in vitro fertilization (IVF) and nuclear transferred (NT)bovine blastocyst. (omitted)