• Reproductive and Developmental Biology
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• 제30권1호
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• pp.47-52
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• 2006

본 연구는 단위발생유래 생쥐 배아줄기세포(P-mES)지가 체외수정유래 생쥐 배아줄기세포 (mES)와 마찬가지로 기능성 심근세포로 체외 분화되는지를 조사하였다. 각 세포주 P-mES04와 MES03를 4일간 부유 배양하여 배아체 (EB)를 형성한 다음 4일간 DMSO를 추가적으로 처리한 뒤 젤라틴이 코팅된 배양접시에 부착시켰다(4-/4+). P-mES04와 mES03으로부터 수축성 심근세포 생성 여부를 30일간 관찰한 결과, 각각 13일(69.83%)과 22일 (61.3%)에 누적 형성율이 가장 높았다. 면역 세포화학염색 결과, 수축성을 나타내는 P-mES04 세포는 수축성 mES03 세포에서와 같이 근육 특이적인 anti-sarcomeric a-actinin 항체와 심근 특이적인 anti-cardiac troponin I 항체에 염색되는 것을 확인하였다. 또한 RT-PCR 결과, 수축성을 나타내는 P-mES04 세포는 심근특이적인 L-type calcium channel, a1C, cardiac myosin heavy chain a, cardiac muscle heavy polypeptide $7{\beta}$, GATA binding protein 4와 atrial natriuretic factor는 발현하나, 골격근 특이적인 L-type calcium channel, a1S는 발현하지 않아 웅성 성체의 심장세포와 유사한 양상을 보였다. 본 연구의 결과는 단위발생 유래 생쥐 배아 줄기세포를 배아줄기세포의 연구의 대체제로 이용할 수 있음을 보여준다.

• 한국가축번식학회지
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• 제24권3호
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• pp.281-288
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• 2000

활성화처리를 위해 22시간 성숙된 난자를 5 $\mu$M ionomycin(I)에서 5분, 10 $\mu$M calcium ionophore(Ca)에서 5분, 2 mM 6-dimethylaminopurine(DMAP)에서 3시간 및 10 $\mu\textrm{g}$/$m\ell$ cycloheximide(CH)에서 6시간 동안 단용 또는 병용 처리하였다. 활성화 처리된 난자는 mCR$_1$aa 배양액 내에서 배양되었으며, 배양조건은 5% $CO_2$, 95% air 또는 5% $CO_2$, 5% $O_2$, 90% $N_2$ 이었다. 1 I, Ca DMAP 및 CH에 의해 처리된 난자의 48시간 난할율은 각각, 12.7%, 14.1%, 28.9% 및 22.9%였다. 그러나, 배반포는 형성되지 않았다. 2. I＋DMAP, I＋CH, Ca＋DMAP및 Ca＋CH에 의해 처리된 난자의 48시간 난할율은 각각, 96.9%, 82.1%, 93.1% 및 84.7%였고, 배반포 발생율은 각각, 10.4%, 5.3%, 17.6% 및 7.1%로, I 및 Ca를 이용하여 세포 내 칼슘수준을 증가시킨 후, DMAP로 3시간 동안 배양하였을 때, 배반포 발생율이 유의적으로 높았다(P<0.05). 3. I, Ca, DMAP 및 CH에 의해 단용 처리된 난자의 전핵 발생율은 각각, 5.4%, 3.6%, 28.3% 및 28.8%였다. 그러나, 병용 처리된 난자는 100%의 전핵 형성율을 나타냈다.

• Reproductive and Developmental Biology
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• 제36권1호
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• pp.33-37
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• 2012

Differential capacity of the parthenogenetic embryonic stem cells (PESCs) is still under controversy and the mechanisms of its neural induction are yet poorly understood. Here we demonstrated neural lineage induction of PESCs by addition of insulin-like growth factor-2 (Igf2), which is an important factor for embryo organ development and a paternally expressed imprinting gene. Murine PESCs were aggregated to embryoid bodies (EBs) by suspension culture under the leukemia inhibitory factor-free condition for 4 days. To test the effect of exogenous Igf2, 30 ng/ml of Igf2 was supplemented to EBs induction medium. Then neural induction was carried out with serum-free medium containing insulin, transferrin, selenium, and fibronectin complex (ITSFn) for 12 days. Normal murine embryonic stem cells derived from fertilized embryos (ESCs) were used as the control group. Neural potential of differentiated PESCs and ESCs were analyzed by immunofluorescent labeling and real-time PCR assay (Nestin, neural progenitor marker; Tuj1, neuronal cell marker; GFAP, glial cell marker). The differentiated cells from both ESC and PESC showed heterogeneous population of Nestin, Tuj1, and GFAP positive cells. In terms of the level of gene expression, PESC showed 4 times higher level of GFAP expression than ESCs. After exposure to Igf2, the expression level of GFAP decreased both in derivatives of PESCs and ESCs. Interestingly, the expression level of $Tuj1$ increased only in ESCs, not in PESCs. The results show that IGF2 is a positive effector for suppressing over-expressed glial differentiation during neural induction of PESCs and for promoting neuronal differentiation of ESCs, while exogenous Igf2 could not accelerate the neuronal differentiation of PESCs. Although exogenous Igf2 promotes neuronal differentiation of normal ESCs, expression of endogenous $Igf2$ may be critical for initiating neuronal differentiation of pluripotent stem cells. The findings may contribute to understanding of the relationship between imprinting mechanism and neural differentiation and its application to neural tissue repair in the future.

• 한국수정란이식학회지
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• 제27권1호
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• pp.57-62
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• 2012

Efficient oocyte activation is a key step for the success of nuclear transfer in cloning. Ionomycin sequentially combined with 6-DMAP is now widely used to activate normal oocytes for analytical studies of oocyte activation and to activate reconstructed oocytes after nuclear transfer. The present study investigated sources of oocytes, duration of ionomycin and 6-DMAP, laser and electric stimulation in goat oocyte activation in order to optimize the protocols. Goat ovaries were collected in individual abattoirs during the breeding season and were delivered to the laboratory within 6 h in saline with 100 IU/ml streptomycin and 0.05 mg/ml penicillin. The oocytes were denuded from the cumulus cell by pipetting with 0.2% hyaluronidase in PBS at 20~22 hr post maturation. Oocytes with the polar body were selected and assigned to four groups for parthenogenetic activation. To examine the effect of duration of ionomycin treatment, oocytes after 20~22 hr of maturation were treated with 2.5 uM ionomycin for 1 or 5 min times and then cultured in 2 mM 6-DMAP for 2 or 4 hr. The activated oocytes were cultured in mSOF at $38.5^{\circ}C$ in $CO_2$ 5%, $O_2$ 5% and $N_2$ 90% multi incubator. Cleavage and blastocyst development was observed at 48 hr and day 8 of culture $in$ $vitro$, respectively. Activation rates of oocytes exposed to ionomycin for 1 min(86.4%) were significantly higher than those treated for 5 min(74.3%) duration. This indicated that 1 min ionomycin treatment was most suitable for activation of goat oocytes. The duration of 6-DMAP treat duration was in 2 mM 6-DMAP for 2 hr after 1 min exposure to 2.5 uM ionomycin. The activation rate of oocytes incubated in 6-DMAP for 2 hour(82.5%) was significantly higher than those in oocytes treated with 4 hr(75.5%).

• 한국수정란이식학회지
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• 제18권1호
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• pp.15-25
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• 2003

발생학에서 키메라(chimera)는 2개 이상의 다른 유전자형의 세포, 또는 다른 종의 세포로부터 만들어진 1개의 생물개체를 뜻하는 말로, 이는 초기 수정란의 발달과 포유류의 분화를 연구하는데 이용되고 있다. 키메라를 만드는 방법에는 할구와 내세포괴를 응집시키는 방법과 배반포 내에 여러 종류의 세포를 주입하는 방법이 있다 본 실험에서는 서로 다른 두 가지 방법의 활성화 처리법, 즉, ionomycin 처리 후 Cycloheximide (CHX) 또는 6-Dimetylaminopurine (6-DMAP)에 따른 단위 발생란의 분할과 단계적인 발달율을 살펴 보고자 하였으며, 서로 다른 방법에 의해 생산된 단위발생란 유래의 할구와 체외수정란 유래 할구를 응집하여 키메라 수정란(chimeric embryo)를 만든 후 체외수정란과 발달율을 비교해 보았다. 도축장 유래의 난소에서 난자를 채취하여 체외에서 22~24시간 성숙시킨 후 난구세포를 제거하고 metaphase II 단계의 난자를 5 $\mu$M ionomycin에 4분간 처리한 후, 10 $\mu$g/ml CHX/5 $\mu$g/ml cytochalasin B (CCB)에 5시간 또는 1.9 mM 6-DMAP에 4시간 처리하여 분할율과 배반포기 발달율을 비교 조사하였다. 난자 분할율에서는 체외수정란과 6-DMAP처리 단위 발생란에서 각각가 83.7 와 85.5%로 CHX/CCB 처리 단위발생란의 57.9%보다 유의적으로 높게(P<0.05) 나타났으며, 배반포기 발달율에 있어서는 체외수정란의 발달율이 27.8%로 6-DMAP처리 활성란 12.3%와 CHX/CCB 처리 활성란 5.3%보다 유의적으로 높게 (P<0.05) 나타났다. 키메라 수정란(chimeric embryo)은 서로 다른 두 가지 처리에 의해 생산된 단위발생란의 할구 2개와 체외수정란 유래의 할구 2개를 빈 투명대 내에서 응집시켜 제조하였다 빈 투명대 내에 키메라 수정란(chimeric embryos)의 8 세포기까지의 발달율은, 체외 수정란 할구 2개와 CHX/CCB 처리에 의한 할구 2개를 응집한 그룹은 46.1%, 체외 수정란 할구와 6-DMAP 유래 할구 2개를 응집한 그룹은 52.8% 였으며, handled control은 54.7%로 체외 수정란 77.7%에 비해 유의적으로 낮게(P<0.05) 나타났다. 배반포기까지의 발달율은 체외 수정란과 CHX/CCB에 의해 생산된 키메라 수정란(chimeric embryo)은 12.8%, 체외 수정란과 6-DMAP에 의한 키메라 수정란(chimeric embryo)은 18.8%로 handled control의 21.4%에 비해 유의적으로 낮았으며(P<0.05), 이들 키메라 수정란(chimeric embryos)은 체외 수정란의 34.9%에 비해 유의적으로 낮게(P<0.05)나타났다. 6-DMAP 처리 단위발생이 유기된 수정란 할구 2개와 체외수정란의 할구 2개의 응집에 의한 키메라 수정란(chimeric embryos)의 발달율이 CHX/CCB와 체외수정란의 응집에 의한 키메라 수정란(chimeric embryos)에 비해 다소 높게 나타났으나, 유의적인 차이는 없었다. 본 실험의 결과 서로 다른 방법에 의한 단위 발생란 유래의 할구와 체외 수정란 유래의 할구가 응집에 의한 재조합이 가능하였고 이들을 체외에서 배양하여 배반포기의 수정란까지 발달시켰다.

• 한국수정란이식학회지
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• 제30권3호
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• pp.219-224
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• 2015

The purpose of this study is to develop transgenic cell line expressing targeted human granulocyte colony stimulating factor (hGCSF) and green fluorescence protein (GFP) genes as well as production of Somatic Cell Nuclear Transfer (SCNT) embryos derived from co-expressed transgenic donor cells. Constructed pPiggy-mWAP-hGCSF-EF1-GFP vector was chemically transfected into bovine fetus cells and then, only GFP expressed cells were selected as donor cells for SCNT. Cleavage and blastocyst rates of parthenogenetic, SCNT embryos using non-TG cell and hGCSF-GFP dual expressed SCNT embryos were examined (cleavage rate: $78.0{\pm}2.8$ vs. $73.1{\pm}3.2$ vs. $70.4{\pm}4.3%$, developmental rate: $27.2{\pm}3.2$ vs. $21.9{\pm}3.1$ vs. $17.0{\pm}2.9%$). Result indicated that cleavage and blastocyst rates of TG embryos were significantly lower (P<0.05) than those of parthenogenetic and non-TG embryos, respectively. In this study, we successfully produced hGCSF-GFP dual expressed SCNT embryos and cryopreserved to produce transgenic cattle for bioreactor system purpose. Further process of our research will transfer of transgenic embryos to recipients and production of hGCSF secreting cattle.

• 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.

• Asian-Australasian Journal of Animal Sciences
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• 제32권8호
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• pp.1112-1121
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• 2019

Objective: Gram-negative bacteria lipopolysaccharide (LPS) has been reported to be associated with uterine impairment, embryonic resorption, ovarian dysfunction, and follicle retardation. Here, we aimed to investigate the toxic effects of LPS on the maturation ability and parthenogenetic developmental competence of bovine oocytes. Methods: First, we developed an in vitro model to study the response of bovine cumulusoocyte complexes (COCs) to LPS stress. After incubating germinal vesicle COCs in $10{\mu}g/mL$ of LPS, we analyzed the following three aspects: the expression levels of the LPS receptor toll-like receptor 4 (TLR4) in COCs, activities of intracellular signaling protein p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-kappa B (NF-${\kappa}B$); and the concentrations of interleukin (IL)-$1{\beta}$, tumor necrosis factor (TNF)-${\alpha}$, and IL-6. Furthermore, we determined the effects of LPS on the maturation ability and parthenogenetic developmental competence of bovine oocytes. Results: The results revealed that LPS treatment significantly elevated TLR4 mRNA and protein expression levels in COCs. Exposure of COCs to LPS also resulted in a marked increase in activity of the intracellular signaling protein p-p38 MAPK and NF-${\kappa}B$. Furthermore, oocytes cultured in maturation medium containing LPS had significantly higher concentrations of the proinflammatory cytokines IL-$1{\beta}$, TNF-${\alpha}$, and IL-6. LPS exposure significantly decreased the first polar body extrusion rate. The cytoplasmic maturation, characterized by polar body extrusion and distribution of peripheral cortical granules, was significantly impaired in LPS-treated oocytes. Moreover, LPS exposure significantly increased intracellular reactive oxygen species levels and the relative mRNA abundance of the antioxidants thioredoxin (Trx), Trx2, and peroxiredoxin 1 in oocytes. Moreover, the early apoptotic rate and the release of cytochrome C were significantly increased in response to LPS. The cleavage, morula, and blastocyst formation rates were significantly lower in parthenogenetically activated oocytes exposed to LPS, while the incidence of apoptotic nuclei in blastocysts was significantly increased. Conclusion: Together, these results provide an underlying mechanism by which LPS impairs maturation potential in bovine oocytes.

• Asian-Australasian Journal of Animal Sciences
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• 제29권10호
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• pp.1500-1507
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• 2016

Myostatin (MSTN) can negatively regulate the growth and development of skeletal muscle, and natural mutations can cause "double-muscling" trait in animals. In order to block the inhibiting effect of MSTN on muscle growth, we transferred zinc-finger nucleases (ZFN) which targeted sheep MSTN gene into cultured fibroblasts. Gene targeted colonies were isolated from transfected fibroblasts by serial dilution culture and screened by sequencing. Two colonies were identified with mono-allele mutation and one colony with bi-allelic deletion. Further, we introduced the MSTN-ZFN mRNA into sheep embryos by microinjection. Thirteen of thirty-seven parthenogenetic embryos were targeted by ZFN, with the efficiency of 35%. Our work established the technical foundation for generation of MSTN gene editing sheep by somatic cloning and microinjection ZFN into embryos.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.198-198
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• 2004

The identification of embryo-specific genes would provide insights into early embryonic development. However, the current methods employed to identify the genes that are expressed at a specific developmental stage are labor intensive and suffer from high rates of false positives. Here we employed a new and accurate reverse transcription-polymerase chain reaction (RT-PCR) technology that involves annealing control primers(ACPs) to identify the genes that are specifically expressed in porcine blastocysts compared to 2-cell stage embryos. (omitted)