• Title/Summary/Keyword: Cloned Embryo

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Cloning and Characterization of Bovine 5-Cytosine DNA Methyltransferase I cDNA

  • Lee, Poongyeon;Min, Kwan-Sik;Lee, Hyun-Gi;Kim, Soon-Jeung;Chung, Hee-Kyoung;Seo, Myung-Kyu;Lee, Yun-Keun;Kim, Sung-Woo;Park, Jin-Ki
    • Proceedings of the KSAR Conference
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    • 2003.06a
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    • pp.39-39
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    • 2003
  • Methylation of DNA 5-cytosine in mammalian early embryo affects great deal in nuclear reprogramming and chromatin remodeling of developing embryo. Current efforts to clone and produce cloned animals including transgenic animals face various problems including low birth rate, irregular development, and so on. In this report, cDNA for the one of house keeping methyltransfcrase, Dnmt1 was cloned from bovine somatic tissues and was analyzed for its nucleotide sequences to investigate the structure and function of the gene in bovine early development. Nucleotide sequence of bovine Dnmt1 homologue showed 76.8% identity with that of human Dnmtl and 66.4% with mouse Dnmt1. Translated amino acid sequence showed 88.4% homology with human homologue and 75.8% homology with mouse counterpart. Three types of Dnmt1 are reported in mouse and human, and are likely present in bovine tissues. Understanding of role of Dnmt1 in bovine development may shed a light in the field of animal, especially bovine cloning.

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Gene Expression of the In Vitro Fertilized or Somatic Cell Nuclear Transfer Embryos Cultured in Medium Supplemented with Different Proteins or Energy Substrates

  • Jang, Goo;Ko, Kyeong-Hee;Jeon, Hyun-Yong;Lee, Byeong-Chun
    • Journal of Embryo Transfer
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    • v.25 no.2
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    • pp.117-125
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    • 2010
  • Several cloned animals have been produced using somatic cell nuclear transfer (SCNT) and have interested in producing the transgenic cloned animals to date. But still its efficiency was low due to a number of reasons, such as sub-optimal culture condition, aberrant gene expression and nuclear reprogramming. The purpose of this study was to analyze gene expression pattern in in vitro fertilized (IVF) or SCNT pre-implantation embryos. IVF- or SCNT-embryos were cultured in media supplemented with different proteins (FBS and BSA) or energy sources (glucose or fructose). Blastocysts from IVF or SCNT were analyzed using semi-quantitative RT-PCR in terms of developmentor metabolic-related genes. Culture medium supplemented different proteins or energy sources had affected on the expression of developmental or metabolic genes in the SCNT blastocysts.

Study on Production of Cloned Animals by Recycling Nuclear Transplantation III. Production of Third Generation Cloned Embryos in Rabbits (반복핵이식에 의한 복제동물 생산에 관한 연구 III. 토끼에서 제3세대 복제수정란의 생산)

  • Lee Hyo-jong;Jeon Byeong-gyun;Yin Xi-jun;Park Choong-saeng;Choe Sang-yong;Yun Chang-hyun;Kang Dae-jin
    • Journal of Veterinary Clinics
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    • v.12 no.1
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    • pp.877-886
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    • 1995
  • The recycling nuclear transplantation(NT) technique has the powerful potential of producing a large number of genetically identical embryos and offsprings from one embryo. Multiple generational cloning by this technique utilizes the NT embryo itself as the donor for the next generation of cloning. In this experiment, we have produced the third generational cloned embryos by recycling NT. Further we examined comparatively the electrofusion rate and in vitro developmental potential in the cloned embryos of the first second and third generations. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulberco's phosphate buffered saline containing 10 % fetal calf serum(FCS) at 47 hours after hCG injection. In the first generation NT, the nuclear donor embryos were synchronized in the phase of Gl/S transition of 32-cell stage. The first and second generation NT embryos developed to 16-cell were used as donor nuclei for second and third generation. The recipient cytoplasms were utilized the oocytes collected at 14 hours after hCG injection, following revoming the nucleus and the first polar body by micromanipulation. The separated blastomeres were injected into the enucleated recipient oocytes by micromanipulation and were fused by electrical stimulation. The electrofusion rate was seen to be 78.0, 88.0 and 90.3 % in the first second and third generation NT rabbit embryos, respectively. The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10 % FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. The in vitro developmental potential to blastocyst stage was significantly(P<0.05) decreased in the third(7.2 %) generation NT embryos compared to the first(53.1 %) and second(16.1 %) generation NT embryos. Following in vitro development to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The mean blastomere numbers and cell cycle numbers of NT embryos during the culture period were significantly(p<0.05) decreased in the second(93.9 cells and 6.55 cylces) and third(81.5 cells and 1.35 cylces) generation, compared to the first(189.9 cells and 7.55 cylces) generation.

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The Suppression of Maturational Competence by Streptomycin during In vitro Maturation of Goat Follicular Oocytes

  • Kang, Jae Ku;Chang, Suk Min;Naruse, Kenji;Han, Jeung Whan;Park, Chang Sik;Jin, Dong Il
    • Asian-Australasian Journal of Animal Sciences
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    • v.17 no.8
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    • pp.1076-1079
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    • 2004
  • Antibiotics are common additives in culture media during in vitro embryo development, but their effects on oocyte maturation in vitro have not been tested. The effects of penicillin, streptomycin and gentamicin on the maturational competence and subsequent development potential of goat follicular oocytes were examined after parthenogenetic activation in vitro. Maturation rates at 24 h after in vitro maturation, and parthenogenetic development at 48 h after activation, were evaluated by observing the protruding first polar body and the 4 cell stage cleavage, respectively. When streptomycin was present in the maturation medium, the percentages of matured oocytes 24 h after activation were significantly (p<0.01) lower than those from the other groups (42.5-45.7% vs. 69.1-73.8%). Penicillin and gentamicin treatment did not affect the maturation rates or the percentages reaching the 4 cell stage 48 h after activation. There was no significant difference in cleavage rates among the different antibiotic treatments 48 h after activation. Therefore, streptomycin suppresses the in vitro maturation of immature goat oocytes, but does not influence their subsequent development.

Epigenetic Reprogramming and Cloning (후성 유전학적 리프로그래밍과 클로닝)

  • Han Yong-Mahn;Kang Yong-Kook;Koo Deog-Bon;Lee Kyung-Kwang
    • Development and Reproduction
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    • v.7 no.2
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    • pp.61-68
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    • 2003
  • Zygote genome should entail a complex process of epigenetic reprogramming including a global DNA demethylation to reach a totipotency or pluripotency during early mammalian development. In this study, we have analyzed methylation patterns in cloned bovine embryos to monitor the epigenetic reprogramming process of donor genomic DNA. Aberrant DNA methylation patterns were observed in various genomic regions of cloned embryos except single-copy gene sequences. The overall genomic methylation status of cloned embryos was quite different from that of normal embryos produced in viかo or in vivo. Abnormal methylation profiles were also specifically represented in trophectoderm cells of cloned embryos, which probably result in widespread gene dysregulation in extraembryonic region or placental dysfunction familiar to cloned animals. Our findings suggest that developmental failures of cloned embryos are due to incomplete epigenetic reprogramming of donor genomic DNA. Understanding the epigenetic reprogramming processes of donor genome will clearly define the faulty development of cloned embryos.

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Improvements in Nuclear Transfer Procedures will Increase Commercial Utilization of Animal Cloning - Review -

  • Stice, S.L.;Gibbons, J.;Rzucidlo, S.J.;Baile, C.A.
    • Asian-Australasian Journal of Animal Sciences
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    • v.13 no.6
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    • pp.856-860
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    • 2000
  • Cloning technology continues to capture widespread attention by the international news media and biomedical and agricultural industries. The future uses of this technology could potentially contribute to major advances in biomedical and agricultural sciences. Cloned transgenic dairy cattle possessing milk promoters directing transgenes will produce pharmaceutical proteins in their milk faster, more efficiently and less expensively than transgenic cattle created using microinjection techniques. Additionally, cloned transgenic fetuses and animals may become a source of cells, tissue and organs for xenotransplantation. Lastly, but maybe most importantly, enhanced production traits and disease resistance may be realized in animal agriculture by utilizing these new technologies. The recent advances in the cattle cloning technology are important but there are still major obstacles preventing widespread commercial use of this technology. The type of donor nucleus, recipient cytoplasm, and cloning procedures used will impact the potential number of clones produced and the uses of the technology. In addition, the new advances in cloning methodology have not improved the relatively low pregnancy rates or reduced the incidence of health problems observed in cloned offspring. These problems may require novel techniques to decipher their cause and new methods of preventing and/or diagnosing them in the preimplantation embryo. The commercial potential is enormous for cloning technology; however, little has been done to improve the efficiencies of the procedure. Improving procedural efficiencies is a critical developmental milestone especially for potential uses of cloning technology in animal agriculture.

Effect of Activation Method and Culture Medium on the Development of Porcine Nuclear Transfer Embryo using Fetal Fibroblast

  • Im, Gi-Sun;Yang, Byoung-Chul;Park, Jin-Ki;Kim, Hyun-Ju;Chang, Won-Kyung;R. S. Prather;B. N. Day
    • Proceedings of the KSAR Conference
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    • 2001.03a
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    • pp.66-66
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    • 2001
  • Since the first birth of pig derived from embryonic cells by nuclear transfer, many researches to produce cloned pig have been carried out. Recently, two reports about the birth of somatic cell cloned pigs using in vivo oocytes and also Betthauser et al. (2000) reported the birth of somatic cell cloned pigs using in vitro oocytes. So here we investigated the effect of activation method and culture medium on in vitro development of porcine nuclear transfer embryo using fetal fibroblast. Oocytes derived from slaughter house obtained ovaries were matured for 42 to 44 h in TCM 199. Matured oocytes were denuded using 0.1% hyaluronidase and then Oocytes with the first polar body were used for enucleation by aspirating the first polar body and adjacent cytoplasm in TCM 199 supplemented with 7.5 $\mu\textrm{g}$ cytochalasin B. Petal fibroblast cells were prepared from 35 days old fetus. To be used as donor cells, fetal fibroblast cells were serum starved for 3 to 5 days and then isolated into single co:1 by trypsinization. Nuclear transfer embryos were fused using 2 times 1.25㎸ for 30$mutextrm{s}$. Fused NT embryos were activated with calcium ionophore (CI) and 6-dimethyl-aminopurine (6-DMAP). Activated oocytes were cultured in NCSU 23 or BECM 3 for 6 days. There was no significant difference between chemical activation and no chemical activation for blastocyst development rate(11.6 vs. 14.8%). However, cell number was significantly higher when NT embryos were activated with CI and 6-DMAP (31.2 vs. 22.6). When NT embryos were cultured in NCSU 23 or BECM 3, blastocyst development rate was 16.4 and 13.2%, respectively, and cell number was 31.5 and 24.1, respectively. These results suggest that chemical activation after fusion and culture in NCSU 23 could increase cell number of porcine NT embryos.

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Expression and Characterization of Bovine DNA Methyltransferase I

  • Chang, Yoo-Min;Yang, Byoung-Chul;Hwang, Seong-Soo;Yoon, Jong-Taek;Min, Kwan-Sik
    • Reproductive and Developmental Biology
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    • v.33 no.2
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    • pp.93-98
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    • 2009
  • In this study, bovine Dnmt1 cDNA was sequenced and detected Dnmt1 mRNA level in bovine tissues by northern blot, methylation pattern of genome by southern blot, specific localization of Dnmt1 in mouse and bovine preimplantation embryos by immunocytostaining and Dnmt1 protein level in ovary and testis by western blot. Bovine Dnmt1 cDNA sequence showed more homology with that of human than mouse and rat. The RNA level of Dnmt1 was 10 times higher expression in placenta than other tissues. This indicates that placenta was hypermethylated compared to others organs. The genomic DNA could not be cut by a specific restriction enzyme (HpaII) in placenta, lung and liver of bovine. It suggests that Dnmt1 in some somatic cells was already methylated. Dnmt1, which has the antibody epitope 1316~1616, was distributed in nucleus and cytoplasm including the stage of pronuclear stage and maturation of oocyte and gradually weaken to blastocyst stage compare to negative. In addition, Dnmt1 was strongly expressed in tetraploid embryo and cloned 8-cell than IVF 8-cell. An aberrant pattern of DNA methylation in cloned embryo may be abnormal development of fetus, embryonic lethality and placenta dysfunction. The somatic specific band (190kDa) was appeared in ovary and testis, but oocyte specific band (175kDa) was not. Further investigations are necessary to understand the complex links between the methyltransferases and the transcriptional activity of genes in the cloned bovine tissues.

Factors Affecting the Efficiency of Animal Cloning by Somatic Cell Nuclear Transfer

  • Kim, Min-Goo;Park, Chi-Hun;Lee, Sang-Goo;Seo, Hee-Won;Choi, Yo-Han;Lee, Chang-Kyu;Ka, Hak-Hyun
    • Journal of Embryo Transfer
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    • v.23 no.2
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    • pp.67-76
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    • 2008
  • Since the birth of Dolly using fully differentiated somatic cells as a nuclear donor, viable clones were generated successfully in many mammalian species. These achievements in animal cloning demonstrate developmental potential of terminally differentiated somatic cells. At the same time, the somatic cell nuclear transfer (SCNT) technique provides the opportunities to study basic and applied biosciences. However, the efficiency generating viable offsprings by SCNT remains extremely low. There are several explanations why cloned embryos cannot fully develop into viable animals and what factors affect developmental potency of reconstructed embryos by the SCNT technique. The most critical and persuasive explanation for inefficiency in SCNT cloning is incomplete genomic reprogramming, such as DNA methylation and histone modification. Numerous studies on genomic reprogramming demonstrated that incorrect DNA methylation and aberrant epigenetic reprogramming are considerably correlated with abnormal development of SCNT cloned embryos even though its mechanism is not fully understood. The SCNT technique is useful in cloning farm animals because pluripotent stem cells are not established in farm animal species. Therapeutic cloning combined with genetic manipulation will help to control various human diseases. Also, the SCNT technique provides a chance to overcome excessive demand for the organs by production of transgenic animals as xenotransplantation resources. Here, we describe the factors affecting the efficiency of generating cloned farm animals by the SCNT technique and discuss future directions of animal cloning by SCNT to improve the cloning efficiency.

Induction of Parturition after Transfer of a Hanwoo-Somatic Cell Cloned Embryo in Holstein Cow (체세포복제수정란 이식에 의한 젖소 수란우의 유도분만)

  • 손동수;서국현;허태영;강석진;류일선;최선호;이장희;박성재;최은주
    • Journal of Embryo Transfer
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    • v.18 no.1
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    • pp.81-84
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    • 2003
  • This study was performed to produce Hanwoo-fetal fibroblast cell clone transferred to Holstein recipient. It was coming on the parturition but didn't show parturition signs until 272 days from the embryo transfer. For the induction of normal parturition, dexamethasone (20 mg) were injected with IM on 272 day and PG $F_{2a}$ (25 mg) plus estradiol (20 mg) was injected with IM after 24hrs. Forty-eight hours after dexamethasone injection, we could find parturition signs and delivered healthy male of offspring (40 kg) about 50 hrs after it's injection. However the recipient was showed retention of afterbirth.h.