• 한국임상수의학회지
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• 제12권1호
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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.

• 한국수정란이식학회지
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• 제23권4호
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• pp.263-267
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• 2008

The purpose of this study was to determine toxic effect of sucrose and trehalose prior to cryopreservation on nuclear maturation and embryonic development in immature bovine oocytes. All cryoprotectant was prepared in tissue culture medium 199-HEPES (TCM 199-HEPES) with 10% fetal bovine serum (FBS). Immature oocytes were exposed to 1.2M ethylene glycol (EG) and 0.1M sucrose or 1.2M EG and 0.1M trehalose for 3 min and then were exposed to 3.2 M EG and 0.25 M sucrose or 3.2 M EG and 0.25 M trehalose for 1 min. Oocytes treated with cryoprotectants were exposed to 0.25 M sucrose or 0.25 M trehalose for 5 min and then 0.1 M sucrose or 0.1 M trehalose for 5 min. Depending on type of sugar added to cryopreservation solution, oocytes were allocated to sucrose group and trehalose group, respectively. Oocytes exposed to TCM 199-HEPES with 10% FBS were considered as control. Oocytes were cultured in TCM 199 supplemented with 10% FBS, 5 ng/ml epidermal growth factor, 0.01 IU/ml luteinizing hormone, and $1\;{\mu}g/ml$ estradiol for 24 h in $39^{\circ}C$, 5% $CO_2$. Nuclear maturation was assessed by staining oocytes with 1% aceto-orcein. Oocytes were fertilized in vitro and were cultured in TCM 199 supplemented with 10% FBS, 5 mM sodium pyruvate, and antibiotics in $39^{\circ}C$, 5% $CO_2$. The rates of cleavage and blastocyst, and cell number in blastocyst were assessed. Metaphase II rates were not different among experimental groups regardless of type of sugar. The cleavage rate of trehalose group (73.3%) was significantly higher (p<0.05) than those of sucrose group (62.8%) and control group (60.8%). The blastocyst rate was significantly higher in trehalose group (p<0.05). Mean cell number in blastocyst were not different among experimental groups, although cell number of blastocyst in trehalose group was significantly higher on day 7 (p<0.05). In conclusion, sucrose and trehalose were not toxic to immature bovine oocytes prior to cryopreservation. In particular, trehalose was more effective on embryonic development.

• Reproductive and Developmental Biology
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• 제37권4호
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• pp.269-279
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• 2013

Low efficiency of somatic cell nuclear transfer (SCNT) is attributed to incomplete reprogramming of transfered nuclei into oocytes. Trichostatin A (TSA), histone deacetylase inhibitor and 5-aza-2'deoxycytidine (5-aza-dC), DNA methylation inhibitor has been used to enhance nuclear reprogramming following SCNT. However, it was not known molecular mechanism by which TSA and 5-aza-dC improve preimplantation embryo and fetal development following SCNT. The present study investigates embryo viability and gene expression of cloned porcine preimplantation embryos in the presence and absence of TSA and 5-aza-dC as compared to embryos produced by parthenogenetic activation. Our results indicated that TSA treatment significantly improved development. However 5-aza-dC did not improve development. Presence of TSA and 5-aza-dC significantly improved total cell number, and also decreased the apoptotic and autophagic index. Three apoptotic-related genes, Bak, Bcl-xL, and Caspase 3 (Casp3), and three autophagic-related genes, ATG6, ATG8, and lysosomal-associated membrane protein 2 (LAMP2), were measured by real time RT-PCR. TSA and 5-aza-dC treatment resulted in high expression of anti-apoptotic gene Bcl-xL and low pro-apoptotic gene Bak expression compared to untreated NT embryos or parthenotes. Furthermore, LC3 protein expression was lower in NT-TSA and NT-5-aza-dC embryos than those of NT and parthenotes. In addition, TSA and 5-aza-dC treated embryos displayed a global acetylated histone H3 at lysine 9 and methylated DNA H3 at lysine 9 profile similar to the parthenogenetic blastocysts. Finally, we determined that several DNA methyltransferase genes Dnmt1, Dnmt3a and Dnmt3b. NT blastocysts showed higher levels Dnmt1 than those of the TSA and 5-aza-dC blastocysts. Dnmt3a is lower in 5-aza-dC than NT, NTTSA and parthenotes. However, Dnmt3b is higher in 5-aza-dC than NT and NTTSA. These results suggest that TSA and 5-aza-dC positively regulates nuclear reprogramming which result in modulation of apoptosis and autophagy related gene expression and then reduce apoptosis and autophagy. In addition, TSA and 5-aza-dC affects the acetylated and methylated status of the H3K9.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.121-121
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• 2003

The standard protocol for the production of transgenic mouse from ES-injected embryo has to process via chimera producing and several times breeding steps, In contrast, tetraploid-ES cell complementation method allows the immediate generation of targeted murine mutants from genetically modified ES cell clones. The advantage of this advanced technique is a simple and efficient without chimeric intermediates. Recently, this method has been significantly improved through the discovery that ES cells derived from hybrid strains support the development of viable ES mice more efficiently than inbred ES cells do. Therefore, the objective of this study was to generate transgenic mice overexpressing human resistin gene by using tetrapioid-ES cell complementation method. Human resistin gene was amplified from human fetal liver cDNA library by PCR and cloned into pCR 2.1 TOPO T-vector and constructed in pCMV-Tag4C vector. Human resistin mammalian expression plasmid was transfected into D3-GL ES cells by lipofectamine 2000, and then after 8~10 days of transfection, the human resistin-expressing cells were selected with G418. In order to produce tetraploid embryos, blastomeres of diploid embryos at the two-cell stage were fused with two times of electric pulse using 60 V 30 $\mu$sec. (fusion rate : 93.5%) and cultured upto the blastocyst stage (development rate : 94.6%). The 15~20 previously G418-selected ES cells were injected into tetraploid blastocysts, and then transferred into the uterus of E2.5d pseudopregnant recipient mice. To investigate the gestation progress, two El9.5d fetus were recovered by Casarean section and one fetus was confirmed to contain human resistin gene by genomic DNA-PCR. Therefore, this finding demonstrates that tetraploid-ES mouse technology can be considered as a useful tool to produce transgenic mouse for the rapid analysis of gene function in vivo.

• Clinical and Experimental Reproductive Medicine
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• 제24권1호
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• pp.51-56
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• 1997

Recently, through the development of the primer extension preamplification(PEP) method which amplifies the whole genome, simultaneous multiple DNA analysis has become possible. Whole genome from each single cell can be amplified using 15 base oligonucleotide random primer. The greatest advantage of PEP-PCR is the ability to investigate several loci simultaneously and confirm results by analysing multiple aliquots for each locus. This technique led to the development of preimplantation genetic disease diagnosis using blastomere from early embryo, sperm, polar body and oocyte. In this study, we applied PEP-PCR in 20 cases of single amniocyte and 20 cases of single chorionic villus cell for the clinical application of the prenatal and preimplantational genetic diagnosis. We analysed 7 gene loci simultaneously which are 46, 47 exons related to dystrophin gene, two VNTR (variable number tandem repeat) markers using 5'dysIII, 3'CA related to dystrophin gene and DYZ1, DYZ3, DYS14 regions on chromosome Y. In all the tests, 97.5% of PEP-PCR amplifications with single cells were successful. We obtained 38/40 (95%) accuracy in gender determination through chromosome analysis comparison. Therefore, these results have significant implications for a sperm or oocyte analysis and prenatal or preimplantational genetic diagnosis.

• Reproductive and Developmental Biology
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• 제29권2호
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• pp.83-91
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• 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.

• 농업과학연구
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• 제33권1호
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• pp.35-41
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• 2006

이종간의 핵이식은 확보가 쉬운 난자를 이용함으로서 용이한 수핵란의 확보와 윤리적인 문제등을 피할 수 있으므로 매우 유용한 방법이다. 본 연구에서는 이러한 이종간 핵이식의 조건을 규명하고자 유산양 태아섬유아세포를 이용하여 돼지의 난자에 이종간 핵이식을 시도하였다. 돼지와 산양의 난소에서 난포란을 채취하여 각각 NCSU-23, TCM-199에 호르몬을 첨가한 성숙배양액에 배양하여 $38^{\circ}C$, 5% $CO_2$의 배양기에서 48시간 동안 체외성숙 시켜 수핵난자를 준비하였다. 공여세포는 유산양의 태아섬유아세포는 DMEM배양액에서 배양한 후 0.25% Trypsin-EDTA 용액으로 처리하여 single-cell로 분리하여 사용하였다. 돼지와 산양의 성숙란에 공여세포를 핵치환하여 0.3 M mannitol fusion medium에 넣어 각각 DC 1.2 kV/cm $30{\mu}sec$과 DC 2.39 kV/cm $15{\mu}sec$의 조건으로 BTX를 이용 2회의 전기충격으로 융합 활성화하였다. 활성화된 돼지와 산양 핵이식란은 각각 PZM-3와 mSOF 배양액에 7일간 배양하면서 할구분열율과 배발달율을 관찰하여 동종간 핵이식란과 비교하였다. 비교결과 이종간 핵이식란의 경우 분열률이 58.9%, 배반포기로의 발달률이 5.4%로 돼지 동종간 핵이식란의 분열율이 67.4%, 배반포기로의 배발달율은 13.6% 보다 낮게 나타났고, 또한 산양의 동종간 핵이식란배아의 분열율 81%, 상실배와 배반포기로의 발달률이 54.7% 보다 낮게 나타났다. 이러한 결과로 이종간의 이식은 많은 잇점에도 불구하고 아직은 낮은 배발달률을 보이고 있어 이에 대한 핵이식 조건 및 체외배양 조건 등 많은 부분에서의 추가적인 연구가 필요한 것으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제20권12호
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• pp.1820-1826
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• 2007

In this study, we conducted various experiments in order to develop enhanced cultural conditions for in vitro-produced porcine embryos. All embryos were produced by in vitro maturation (IVM) and fertilization (IVF) of immature oocytes from abattoir-derived ovaries. In experiment 1, we cultured IVF embryos in 4 different groups, namely, 0% bovine serum albumin (BSA), 3% BSA, 0.05% Polyvinyl alcohol (PVA), and 0.5% Polyvinylpyrrolidone (PVP) added to the basal fluid cultural medium, Porcine zygote medium 3 (PZM-3). The rates of embryo development were higher in the group where the PZM-3 media had been supplemented with 3% BSA than the other groups. While not statistically significant, the percent of blastocysts and hatched blastocytes were 6.9% and 25.0% in the 3% BSA group vs. 1.2-6.4% and 0-16.7% in the other groups, respectively. In experiment 2, we added 10% fetal bovine serum (FBS) to PZM-3 on day 0 of culture and observed the development rate of blastocysts per day of culture from days 0 to 5. The development rate of blastocysts was higher at 15.6% on day 4 than on any other day, and was significantly higher than on day 0 or day 1 (p<0.05). The development rate of hatched blastocysts was 26.7% on day 4, and was higher than on any other day. In experiment 3, we cultured IVF embryos with different fluid culture media, grouped as 1) PZM-3+0.3% BSA (day0-day7); 2) PZM-3+0.3% BSA${\rightarrow}$day-4) PZM-3+10% FBS; 3) PZM-3+0.3% BSA${\rightarrow}$PZM-3+0.3% BSA+(day-4) FBS 10%; and 4) PZM-3+0.3% BSA+10% FBS (day0-day7). The development rates of blastocysts and hatched blastocysts were 21.5% and 53.1% in group 3, respectively, which was significantly higher than group 4 with respect to blastocyst development (5.2%, p<0.05) but not hatched blastocysts (14.3%). The total cell number (TCN) of blastocysts in group 3 was higher at $37.8{\pm}16.1$ than the other groups at $16.8{\pm}4.4$ - $30.1{\pm}10.9$; however, this was not significantly different. The results of this study showed that PZM-3 containing 0.3% BSA and supplemented with FBS during the later stage of culture on day 4 resulted in better TCNs and an increased rate of hatched blastocysts.

• 한국가축번식학회지
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• 제27권1호
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• pp.87-93
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• 2003

칡소의 귀세포를 이용한 핵이식 시에 전기 융합조건이 핵이식란의 발생에 미치는 영향을 알아보고, 칡소의 귀세포와 한우의 태아섬유아세포를 이용한 핵이식란을 이식하여 수태율을 비교하였다. 전기자극 조건에 따른 핵이식 후 융합율은 51∼68%의 범위로 차이는 없었으나, lysis 율은 1.9kv/cm의 10us, 20us에서 각각 0.0과 38.7%, 2.0kv/cm의 10us, 20us에서 각각 2.9와 37.5%, 2.1kv/cm의 10us, 20us에서 각각 21.2와 51.8%을 보였다. 난할율은 1.9kv/cm의 10us, 20us에서 각각 75.8과 69.8%, 2.0kv/cm의 10us, 20us에서 각각 76.9와 68.8%, 2.1kv/cm의 10us, 20us에서 각각 70.5와 68.5로 큰 차이가 없었다. 그러나 배반포 발생율은 1.9kv/cm의 10us, 20us 각각 19.5와 48.6%, 2.0kv/cm의 10us, 20us에서 각각 20.0과 40.9%, 2.1kv/cm의 10us, 20us에서 각각 44.2와 27.0%로 각 조건에서 시간에 따른 차이를 보였다. 핵이식에서 공여세포와 세포질간의 융합율과 lysis율은 전압과 통전시간에 영향을 받으며 전압을 높이는 것이 통전시간을 길게 하는 경우보다 수핵난자의 세포질에 상해를 줄이고 배반포 발생에 유리 하였다. 한우의 태아섬유아세포의 핵이식후 생산된 배반포를 5마리의 수란우에 이식한 결과 두 마리가 임신되었고(40.0%), 칡소의 귀 세포를 핵이식한 후 생산된 배반포를 19마리에 이식한 결과 3마리가 임신되었다(15.8%).

• Clinical and Experimental Reproductive Medicine
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• 제39권3호
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• pp.118-124
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• 2012

Objective: To review the outcomes of preimplantation genetic diagnosis (PGD) using zona drilling with acid Tyrode's solution (chemical zona pellucida drilling, chemical ZD) and those of partial zona dissection (PZD). Methods: Clinical outcomes of seventy-one couples undergoing 85 PGD cycles from January 2005 to December 2010 were included. Blastocyst formation and the hatching rate, clinical pregnancy rate, ongoing pregnancy rate, implantation rate, and fetal gender ratio of the PZD and chemical ZD groups were compared. Results: Application of PZD resulted in a significantly higher rate of clinical pregnancy (40.7% vs. 15.4%, p=0.022), ongoing pregnancy (35.6% vs. 11.5%, p=0.023), and implantation (18.1% vs. 5.7%, p=0.007) compared with chemical ZD. Among non-transferred embryos, the rate of blastocyst formation on day 5 (49.1% vs. 39.5%, p=0.016) and hatching on day 6 (47.2% vs. 26.5%, p<0.001) were also significantly higher in the PZD group. Conclusion: The mechanical zona dissection (ZD) method showed better outcomes than chemical ZD in terms of the blastocyst development and pregnancy rate. In this study, the fact that chemical ZD was conducted in different period from mechanical method should be considered in interpreting the result.