• Journal of Animal Science and Technology
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• v.59 no.11
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• pp.24.1-24.14
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• 2017

Over the past decades, in vitro culture media have been developed to successfully support IVF embryo growth in a variety of species. Advanced reproductive technologies, such as somatic cell nuclear transfer (SCNT), challenge us with a new type of embryo, with special nutritional requirements and altered physiology under in vitro conditions. Numerous studies have successfully reconstructed cloned embryos of domestic animals for biomedical research and livestock production. However, studies evaluating suitable culture conditions for SCNT embryos in wildlife species are scarce (for both intra- and interspecies SCNT). Most of the existing studies derive from previous IVF work done in conventional domestic species. Extrapolation to non-domestic species presents significant challenges since we lack information on reproductive processes and embryo development in most wildlife species. Given the challenges in adapting culture media and conditions from IVF to SCNT embryos, developmental competence of SCNT embryos remains low. This review summarizes research efforts to tailor culture media to SCNT embryos and explore the different outcomes in diverse species. It will also consider how these culture media protocols have been extrapolated to wildlife species, most particularly using SCNT as a cutting-edge technical resource to assist in the preservation of endangered species.

• Reproductive and Developmental Biology
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• v.29 no.4
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• pp.229-234
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• 2005

Methods for activation of reconstructed oocytes were examined for the production of nuclear transfer (NT) rat embryos using fetal neural stem cells as donor. Neural stem cells were isolated from Day 14.5 rat fetuses, and the oocytes for recipient cytoplasm were recovered from 4-week old Sprague Dawley rats. After enucleation and nuclear injection, the reconstructed oocytes were immediately exposed to activation medium consisting of 10 mM $SrCl_2$ for 4 h (immediate activation after injection; IAI), or cultured in vitro for $2\~3$ h before activation treatment (injection before activation; IBA). Pre-activated oocytes were also used for NT to test reprogramming potential of artificially activated oocytes. The oocytes were grouped as IIA (immediate injection after activation) and ABI (activation $2\~3$ h before injection). Following NT, the oocytes were cultured in vitro. Development of the NT embryos was monitored at 44 and 119 h after activation. The embryos in groups IAI, mA, and IIA were cleaved to the 2-cell stage at the rates of $36.6\%\;(15/41),\;39.5\%\;(17/43)\;and\;46.3\%$ (25/54), respectively. However, in the ABI group, only one embryo ($1.8\%$, 1/55) was cleaved after activation. After in vitro culture, two NT embryos from IAI group had developed to the morula stage $(4.9\%\cdot2/41)$. However, no morula or blastocyst was obtained in the other groups. These results suggest that immediate activation after injection (IAI) method may be used for the production of rat somatic cell NT embryos.

• Korean Journal of Animal Reproduction
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• v.26 no.4
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• pp.329-338
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• 2002

This study was carried out to investigate the developmental rates of embryo reconstructed with different cell type and to estimate correlation of transcriptional level of octamer-binding transcription factor 4 (Oct4) and fibroblast growth factor 4 (FCF4) gene on peri-implantation stage embryos. Donor cells were transferred into perivitelline space of enucleated oocytes. The karyoplast-cytoplast couplets were accom- plished by cell to cell fusion and activated with ionomycin and 6-dimethylaminopurine. Reconstructed embryos were co-cultured with bovine oviduct epithelial cells in CR 1 aa medium. There is no difference in blastocyst formation rate following nuclear transfer UT) with fetal fibroblast cell (16/50; 32.0%), cumulus cell (16/49; 32.6%) and ear cell (17/52; 32.6%). The expression level of Oct4 and FCF4 in peri-implantation bovine embryo derived from in vitro fertilization (IVF) and NT were determined by reverse-transcription polymerase chain reaction (RT-PCR) technique. In peri-implantation of IVF result in a transient increased of FCF4 paralleled by an increased expression of Oct4. However, Oct4 gene was highly expressed in hatching blastocysts derived from NT compared to IVF. Also, FGF4 expression level in hatching blastocysts and outgrowth stage derived from NT was lower than that of IVF. In conclusion, it is suggested that the different transcription patterns observed in nuclear transfer embryos may lead to a lower rate of embryo development, implantation and pregnancy.

• Korean Journal of Animal Reproduction
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• v.24 no.4
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• pp.429-437
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• 2000

This study investigated the successful introduction of genes of erythropoietin (EPO) and enhanced green fluorescent protein (EGFP) in bovine embryos following nuclear transfer of bovine fetal fibroblasts (bFF), which were transfected by retrovirus vector system. Non-starved bFF were, transferred into perivitelline space of enucleated oocytes. The bFF-oocyte units were accomplished by cell to cell fusion and activated with calcium inophore and 6-dimethylaminopurine. Reconstructed embryos were co-cultured with bovine oviduct epithelial cells in CRlaa medium for 8 days. Out of 187 (EPO) and 210 (EGFP) bovine eggs reconstructed by nuclear transfer, 149 (EPO : 80.0%) and 158 (EGFP : 75.2%) embryos were cleaved, and among them 36 (EPO : 24.2%) and 35 (EGFP : 22.2%) embryos developed to the blastocyst stage. Of these blastocysts, 100% integration of EPO gene in 36 embryos was determined by PCR, and 100% expression of EGFP gene in 35 embryos was observed under the fluorescent microscope. This result indicates that bovine oocytes reconstructed by nuclear transfer of transfected bFF can successfully develop to the blastocyst stage. Furthermore, this novel procedure may be presumably an attractive method efficiently to produce the transgenic cattles.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.136-136
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• 2003

Mainly due to deficiencies in nuclear reprogramming, gene expression and DNA fragmentation, which result in early and late embryonic losses, the overall success rate achieved by cloning techniques to date is low. This present study compared the incidences of DNA fragmentation during development of IVF, parthenotes (PT), nuclear transfer (NT) and transgenic (TG) embryos. Terminal deoxynucleotidyl transferase (TdT) nick-end labelling (TUNEL) with propidium iodide counter staining was used for determination of DNA fragmentation and total number, respectively. TG and NT donor cells were fetal fibroblasts with or without transfection with EGFP, and cultured in DMEM+15% FCS until confluent, for 5 days. At 19 h post-maturation (hpm), enucleated oocytes were reconstructed with donor cells and activated at 24 hpm with the combinations of ionomycin (5 M, 5 min) and cyclo-heximide (10 g/ml, 5 h) after electric fusion by a single DC pulse (1.6 KV/cm, 60 sec). Parthenotes were produced by the same activation protocol at 24 hpm. (중략)

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.5
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• pp.718-724
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• 2007

Considerable attention has been focused on the cloning of mammalian embryos, as a consequence of poor development, in order to enhance the application of genetic engineering. Experiments were conducted to compare the developmental competence of parthenotes and reconstructed (NT) rabbit eggs with fetal fibroblasts (FFs) following various activation regimens. Oocytes and NT eggs were exposed to: electric stimulation (EST, Group 1) and EST followed by 6-dimethylaminopurine (DMAP, Group 2), cycloheximide (CHX, Group 3) or DMAP/CHX (Group 4). Pronuclear (PN) status, cleavage, blastocyst development and the ploidy were assessed. In parthenote groups 1, 2, 3 and 4, the PN formation differed significantly. And, the cleavage and blastocyst rates were 41.7 and 5%, 75.6 and 53.7%, 68 and 36%, 82.1 and 52.6%, respectively, among treatments. Polyploidy was observed in 17.2% of EST plus DMAP and 44.9% of EST plus DMAP/CHX groups. In SCNT groups (Group 1, 2, 3 and 4), the cleavage and blastocyst rates were 28.6 and 7.1%, 58.3 and 29.2%, 56.8 and 24.1%, 64.5 and 27.8%, respectively. The chromosomal composition differed significantly (p<0.05) among treatments. In Group 2 and 3, 53.8% and 81.8% of embryos revealed diploid chromosomal sets, respectively. However, in Group 4, 53.3% of embryos showed abnormal ploidy (mixoploid). Although DMAP or combination with DMAP/CHX resulted in higher in vitro development of rabbit SCNT embryos, higher incidence of chromosomal abnormality may induce problems related to fetal loss of at late stage of development.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.11-11
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• 2002

Interspecies nuclear transfer has been interested to determine ability of oocyte cytoplasm to support reprogramming of somatic cell nuclei of different species. In this study, we investigated developmental ability and mRNA expression patterns of developmentally important genes in bovine reconstructed embryos using a mouse fibroblast cell nucleus. While 20% nuclear transferred embryos with bovine fibroblast developed to morulae/blastocysts, a few(2-5%) nuclear transferred bovine embryos with mouse fibroblast developed to morula. (omitted)

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.48-48
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• 2002

Chimerism has become an important tool for investigating fundamental aspects of early embryonic development and differentiation in mammals for producing transgenic animals. The objective of this study was to evaluate the developmental capacity of chimeric embryos reconstructed with parthenotes and IVF bovine embryos into empty zona pellucida. The MII oocytes were activated by two treatment groups [Group 1, 5 μM inomycin, 5min, ＋ 10 ㎍/㎖ cycloheximide (CHX)/5 ㎍/㎖ cytochalasin B (CCB), 3 h; Group 2, 5 μM ionomycin, 5 min ＋ 1.9 mM 6-dimetylaminopurine (6-DMAP), 3 h]. (omitted)

• Korean Journal of Animal Reproduction
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• v.24 no.4
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• pp.407-417
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• 2000

The present study was to examine effects of various electrical stimulus treatments used for electro-fusion on the preimplantation development of bovine nuclear transfer (NT) embryos with fetal fibroblast cells. Fetal fibroblast cells were isolated from one fetus at day 45 of gestation in Holstein cow, and passaged 3 to 4 times before being transferred into enucleated oocytes. Single fibroblast cells were individually placed into the perivitelline space of enucleated oocytes by using a micromanipulator. At first, the fusion and developmental rates of reconstructed oocytes were compared between different electric stimulation conditions. When fusion of the reconstructed oocyte was induced by different electric pulse periods (15, 30 and 45 $\mu$sec) at a DC pulse of 1.8 kV/cm, 15 (45.5%, 120/264) or 30 $\mu$ sec group (43.9%, 106/241) showed a higher fusion rate than 45 $\mu$sec group (23.2%, 58/250, P＜0.05). However, no difference was detected in the development rate of the fused oocytes to blastocysts between groups. Next experiment was to examine the effects of different electrical field strengths (1.5, 1.8 and 2.1 kV/cm) for 15 $\mu$sec at electrofusion on in vitro development of the NT embryos. As results, there was no difference in the fusion and developmental rates of the NT embryos between electrical strength (P＞0.05). Finally, developmental competence of bovine NT embryos with somatic cells was compared with IVF-derived embryos. Of enucleated oocytes fused with fibroblast cells, 27.4% (75/274) developed to the blastocyst stage, which is similar to that (24.5%, 58/237) of IVF-derived embryos. However, mean nuclei number of NT blastocysts was smaller than that of IVF-derived blastocysts. Thus, we have established an optimal condition (1.8 kV/cm, 15 $\mu$sec) for electric fusion of bovine NT oocytes with somatic cells. The present study indicates that bovine reconstructed embryos with somatic cells normally develop to blastocyst stage in vitro, although having smaller nuclei numbers of blastocysts as compared to IVF-derived embryos.

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