• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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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.

• 한국수정란이식학회지
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• 제29권2호
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• pp.119-125
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• 2014

Although the majority of surviving pigs cloned by somatic cell nuclear transfer (SCNT) appear to be physiologically normal, there is a general lack of detailed hemato-physiologic studies for the period of early adulthood to substantiate this claim. In the present study, we investigated variation in blood chemistry and endocrinological parameters between mesenchymal stem cells (MSCs) derived from cloned and normal age-matched female and male miniature pigs. Cloned females and males showed normal ranges for complete blood count assessments. Biochemical assessments showed that ${\gamma}$-GGT, ALT and cholesterol levels of male and female clones were significantly (P<0.05 or P<0.01, respectively) higher than that of age-matched control miniature pigs. Variations in insulin and IGF-1 were higher in female clones than in male clones and controls. Thus, although female and male cloned miniature pigs may be physiologically similar to normal animals, or at least within normal ranges, a greater degree of physiological and endocrinological variation was found in cloned pigs. The above variation must be taken into account before considering cloned female or male miniature pigs for various biomedical applications.

• Asian-Australasian Journal of Animal Sciences
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• 제21권8호
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• pp.1127-1133
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• 2008

This study was designed to develop a method of liquid storage of boar sperm at $4^{\circ}C$ by using the modified Beltsville F5 (BF5) diluent with bovine serum albumin (BSA) and N-acetyl-D-glucosamine. Boar sperm were stored in lactose-egg yolk and N-acetyl-D-glucosamine (LEN), BF5 and Golden-Pig liquid 4 (GPL4) diluents at $4^{\circ}C$ for 5 days and were examined for sperm viability, adenosine triphosphate (ATP) and in vitro fertilization (IVF). The percentage of sperm viability in GPL4 diluent was higher than in LEN and BF5 diluent from 1 to 5 days of storage at $4^{\circ}C$. The percentage of sperm viability steadily declined from 1 to 5 days of storage in the three different diluents. Sperm ATP in GPL4 diluent was higher than in LEN and BF5 diluents from 1 to 5 days of storage. Sperm ATP rapidly declined after 5 days of storage in the three different diluents. Porcine oocytes matured in vitro were inseminated with different sperm concentrations of liquid semen stored for 3 days in GPL4 diluent. The percentage of monospermic oocytes did not show any differences from 2.5 to $20{\times}10^5$ sperm/ml. However, the percentage of polyspermic oocytes in the sperm concentration of $2.5{\times}10^5$ sperm/ml was lower than in concentrations of 5, 10 and $20{\times}10^5$5 sperm/ml. The percentage of blastocysts from the cleaved oocytes at $2.5{\times}10^5/ml$ sperm concentration was significantly lower than at 5, 10 and $20{\times}10^5sperm/ml$ concentrations. In conclusion, GPL4 diluent can be stored at $4^{\circ}C$ for 5 days and showed higher sperm viability and sperm ATP concentration compared with LEN and BF5 diluents. Also, we found that GPL4 diluent can be used for IVF of porcine oocytes.

• Reproductive and Developmental Biology
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• 제31권2호
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• pp.97-102
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• 2007

The objective of this study was to analyzed pattern of proteins expression abnormally in cloned bovine placenta. TIMP-2 protein whose function is related to extracellular matrix degradation and tissue remodeling processes was one of differentially up-regulated proteins in SCNT placenta. And one of down-regulated protein in SCNT placenta was identified as vimentin protein that is presumed to stabilize the architecture of the cytoplasm. The expression patterns of these proteins were validated by Western blotting. To evaluate how regulatory loci. of TIMP-2 and vimentin genes was programmed reprogramming in cloned placenta. the status of DNA methylation in the promoter region of TIMP-2 and vimentin genes was analyzed by sodium Bisulfite mapping. The DNA methylation results showed that there was not difference in methylation pattern of TIMP-2 and vimentin loci between cloned and normal placenta. Histone H3 acetylation state of the nucleosome was analyzed in the cloned placental and normal placenta by Western blotting. A small portion of the protein lysates were subjected to Western blotting with the antibodies against anti acetyl-Histone H3. Overall histone H3 acetylation state of SCNT placenta was significantly higher than those of normal placenta cells. It is postulated that cloned placenta at the end of gestation seems to be unusual in function and morphology of placenta via improper expression of TIMP-2 and vimentin by abnormal acetylation states of cloned genome.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.18-18
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• 2003

In human, sudden infant death syndrome(SIDS) is synonyms for the sudden, unexpected and unexplained death of an infant. The incidence of SIDS has been estimated to be from 1 to 3%. Cloning has a relatively high rate of late abortion and early postnatal death, particularly when somatic cells are used as donors of nuclei and rates as high as 40 to 70% have been reported. However, the mechanisms for SIDS in cloned animals are not known yet. To date, few reports provide detailed information regarding phenotypic abnormality of cloned pigs. In this study, most of the cloned piglets were alive at term and readily recovered respiration. However, approximately 82% of male cloned piglets (81/22) died within a week after birth. Significant findings from histological examinations showed that 42% of somatic cloned male piglets died earlier than somatic cloned female piglets, most probably due to severe congestion of lung and liver or neutrophilic inflammation in brain, which indicates that unexpected phenotypes can appear as a result of somatic cell cloning. No anatomical defects in cloned female piglets were detected, but three of the piglets had died by diarrhea due to bacterial infection within 15 days after birth. Although most of male cloned piglets can be born normal in terms of gross anatomy, they develop phenotypic anomalies that include leydig cell hypoplasia and growth retardation post-delivery under adverse fetal environment and depigmentation of hair- and skin-color form puberty onset. This may provide a mechanism for development of multiple organ system failure in some cloned piglets. Th birth weights of male cloned pig in comparison with those of female cloned piglets are significantly reduced(0.8 vs 1.4kg) and showed longer gestational day(120 vs 114). In conclusion, brain meningitis and hepatopneumonic congestion are a major risk factor for SIDS and such pregnancy in cloned animals requires close and intensive antenatal monitoring.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2001년도 발생공학 국제심포지움 및 학술대회 발표자료집
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• pp.25-31
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• 2001

• 한국가축번식학회지
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• 제20권3호
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• pp.323-331
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• 1996

포유동물에 있어서 조기 성 판정기술은 축산에 있어서의 성별 육종프로그램이나 인간의 X-염색체 관련 열성유전병의 산전진단 등 여러 분야에 응용될 수 있다. 초기배에 대한 성 판정은 성염색체에 존재하는 특이한 염기서열을 증폭시키는 polymerase chain reaction (PCR)과 X와 Y 염색체에 대한 특이적 probe를 이용하는 fluorescent in situ hybridization (FISH)에 의하여 수행될 수 있다. 1992년과 93년, 2개년도에 걸쳐 본 연구실에서 돼지의 3.3 kb 웅성특이 DNA 절편(pEM39)을 cloning하였다. 본 연구는 pEM39가 성특이 DNA-probe로 이용될 수 있는지를 조사하기 위해 PCR과 FISH를 이용하였다. 돼지 난자는 도축장에서 구입한 돼지 난소로부터 채취되었고, 체외배양후 체외수정되었다. 한편 처녀발생나자를 negative control로 이용하였다. 2 세포기의 수정란을 선발한 후 PCR을 통하여 DNA를 분석한 결과, 10개의 수정란 중 6개는 자성, 다른 4개는 웅성으로 판정되었으며, FISH를 수행한 결과, done된 웅성특이 DNA 단편은 돼지 간조직과 초기배에서 웅성특이성을 보였다. 또한 FISH와 karyotyping을 수행한 결과 clone된 웅성특이 DNA 단편이 Y 염색체 q-arm의 heterochromatic region에 위치함을 알 수 있었다. 이러한 결과로 보아 clone된 웅성특이 DNA 단편이 초기배의 성을 조기판정하는데 있어 유용하리라 사료되며, PCR에 의한 초기배의 성 판정에 있어 신뢰할만할 지표가 될 수 있을 것이다.

• 한국수정란이식학회지
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• 제26권1호
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• pp.79-84
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• 2011

This study was performed to identify the differentially methylated region (DMR) and to examine the mRNA expression of the imprinted H19 gene in day 35 of SCNT pig fetuses. The fetus and placenta at day 35 of gestation fetuses after natural mating (Control) or of cloned pig by somatic cell nuclear transfer (SCNT) were isolated from a uterus. To investigate the mRNA expression and methylation patterns of H19 gene, tissues from fetal liver and placenta including endometrial and extraembryonic tissues were collected. The mRNA expression was evaluated by real-time PCR and methylation pattern was analyzed by bisulfite sequencing method. Bisulfite analyses demonstrated that the differentially methylated region (DMR) was located between -1694 bp to -1338 bp upstream from translation start site of the H19 gene. H19 DMR (-1694 bp to -1338 bp) exhibits a normal mono allelic methylation pattern, and heavily methylated in sperm, but not in oocyte. In contrast to these finding, the analysis of the endometrium and/or extraembryonic tissues from SCNT embryos revealed a complex methylation pattern. The DNA methylation status of DMR Region In porcine H19 gene upstream was hypo methylated in SCNT tissues but hypermethylated in control tissues. Furthermore, the mRNA expression of H19 gene in liver, endometrium, and extraembryonic tissues was significantly higher in SCNT than those of control (p<0.05). These results suggest that the aberrant mRNA expression and the abnormal methylation pattern of imprinted H19 gene might be closely related to the inadequate fetal development of a cloned fetus, contributing to the low efficiency of genomic reprogramming.

• Journal of Animal Science and Technology
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• 제63권2호
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• pp.281-294
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• 2021

Although somatic cell nuclear transfer (SCNT) is frequently employed to produce cloned animals in laboratories, this technique is expensive and inefficient. Therefore, the handmade cloning (HMC) technique has been suggested to simplify and advance the cloning process, however, HMC wastes many oocytes and leads to mitochondrial heteroplasmy. To solve these problems, we propose a modified handmade cloning (mHMC) technique that uses simple laboratory equipment, i.e., a Pasteur pipette and an alcohol lamp, applying it to porcine embryo cloning. To validate the application of mHMC to pig cloning, embryos produced through SCNT and mHMC are compared using multiple methods, such as enucleation efficiency, oxidative stress, embryo developmental competence, and gene expression. The results show no significant differences between techniques except in the enucleation efficiency. The 8-cell and 16-cell embryo developmental competence and Oct4 expression levels exhibit significant differences. However, the blastocyst rate is not significantly different between mHMC and SCNT. This study verifies that cloned embryos derived from the two techniques exhibit similar generation and developmental competence. Thus, we suggest that mHMC could replace SCNT for simpler and cheaper porcine cloning.

• Journal of Animal Science and Technology
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• 제50권5호
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• pp.641-648
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• 2008

체세포핵이식을 이용하여 복제동물을 생산하고자하는 연구가 계속적으로 진행되고 있으며 현재까지 많은 성과를 거두고 있으나 복제동물의 성공률이 현저히 낮은 것은 잘 알려진 사실이다. 본 연구에서는 복제동물생산과 관련된 여러 가지 이유 중 정상태반과 체세포복제소의 태반사이의 차등발현 유전자를 발굴하기 위해서 각각의 태반에서 total RNA를 추출하였고 이를 20개의 arbitrary ACPs를 이용하여 정상과 체세포이식태반사이에서 차등발현되는 유전자를 조사한 결과 8개의 발현차이를 보이는 band를 확인할 수 있었고 이 유전자들의 염기서열을 이용하여 BLAST search한 결과 정상태반에서는 CTSZ, LOC509426 및 ELF1 유전자의 발현이 높았고 체세포이식태반에서는 TIMP2, PAG1B, PAG-21, LOC782894, SERPINB6 및 mKIAA2025 protein 유전자의 발현이 높아 총 9개의 차등발현 유전자를 확인할 수 있었다. 이 결과중 체세포핵이식태반에서 발현이 높은 유전자중 아직까지 기능이 밝혀지지 않은 mKIAA 2025 protein를 제외하고 5개의 유전자는 quantitative real-time PCR를 이용하여 유전자의 발현을 재확인하여 체세포핵이식태반에서 발현이 높음을 재확인하였다. 본 연구는 체세포핵이식태반에서 발현차이를 보이는 유전자들 중 극히 일부분만을 확인하였으나 앞으로 더 많은 유전자들과 상호관계를 확인한다면 체세포복제생산에서 태반내의 유전자 변화에 관한 기전을 밝히는데 도움이 될 것이라고 사료된다.