• 생명과학회지
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• 제16권4호
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• pp.566-570
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• 2006

현재, 유전자의 in vivo 기능을 연구하기 위해 가장 많이 이용되고 있는 transgenic mice를 생산하기 위한 기본적으로 이용되고 있는 방법이 one cell-stage embryo에 pronuclear microinjection (PMI)이다. 그러나, 이 PMI 후에 one cell-stage embryo들의 생존율은 현저히 감소 (65.4%)할 뿐만 아니라 PMI 후의 embryo의 출생률(26.4%)이 PMI 처리를 하지 않은 것 (41.9%) 보다 현저히 낮다. 더욱이, PMI 방법에 의해 태어난 transgenic founder들의 간 조직에 병리학적 변화가 14.8% 정도에 대해서 같은 한배의 새끼 non-transgenic founder들의 경우도 간 조직에 병리학적 변화가 14.3%로 나타났다. 결론적으로, 이 PMI 방법에 의한 염색체에 물리적 손상은 형질전환 마우스의 생산 및 표현형에 영향을 미치는 잠재적 요소로 생각된다.

• 한국가축번식학회지
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• 제17권3호
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• pp.193-199
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• 1993

Bovine follicular oocytes were matured in two different conditions, TCM 199＋10% FBS with or without hormones (0.01 unit/ml ovine follicle stimulating hormone, 0.01 unit/ml ovine luteinizing hormone and 1$\mu\textrm{g}$/ml $\beta$-estradiol). There was no significant difference in maturation and fertilization rates of the oocytes between two groups. The result indicates that hormonal treatment does not have beneficial effect on in vitro maturation and fertilization of follicular oocytes. IVF-derived cone-cell bovine embryos were injected with foreign DNA (CChcLf) by microinjection method and then co-cultrued with bovine oviductal epithelial cells. Developmental rate of microinjected embryos to blastocyst stage (21%) was similar to that of non-injected embryos(29%). This result represents that microinjected bovine embryos produced in vitro have a potential of development to normal blastocysts.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.18-18
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• 2001

Despite of importance of integrated events of nucleus and microtubule remodeling in nuclear transferred embryos with somatic cells, little information is available on this subject. In this study we configured chromatin and microtubule organization following somatic cell nuclear transfer in pre- and non-activated bovine oocytes in order to clearify nuclear remodeling process and to demonstrate centrosome inheritance during nuclear transfer. The cumulus-oocyte complexes were collected from slaughterhouse and were matured in vitro for 20 h in TCM 199 supplemented hormone. Matured bovine oocytes were enucleated by aspirating the frist polar body and metaphase chromatin using a beveled pipette. Bovine fibroblast cells were fused into enucleated oocyte by electrical stimulation. Reconstructed oocytes were activated with ionomycine and 6-dimethylaminopurin, and then cultured in CRlaa medium. The organization of nuclear and microtubules were observed using laser-scanning confocal microscopy. At 1 hour after fusion, microtubule aster was seen near the transferred nucleus in most oocytes regardless activation condition. While most of fibroblast nuclei remodeled to premature chromosome condensation (PCC) and to the two masses of chromosome in non-activated oocytes, a few number of fibloblasts went to PCC and multiple pronuclear like structures in activated oocytes. Microtubular spindle was seen around condensed chromosome. Gamma-tubulin was detected in the vicinity of condensed chromosome, suggesting this is a transient spindle. The spindle seperated nucleus into two masses of chromatin which developed to the pronuclear like structures. Two pronuclear like structures were than apposed by microtubular aster and formed one syngamy like nuclear structure at 15 h following nuclear transfer. At 17 to 18 h after fusion, two centrosomes were seen near the nucleus, which nucleates micrtubules for two cell cleavage. While 31% of reconstructed oocytes in non-activated condition developed to morulae and blastocysts, a few reconstructed oocytes in pre-activated condition developed to the blastocyst. These results suggested introduction of foreign centrosome during nuclear transfer, which appeared to give an important role for somatic cell nuclear reprogramming.

• Asian-Australasian Journal of Animal Sciences
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• 제30권7호
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• pp.944-949
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• 2017

Objective: Investigated the effect and mechanism of ascorbic acid on the development of porcine embryos produced by somatic cell nuclear transfer (SCNT). Methods: Porcine embryos were produced by SCNT and cultured in the presence or absence of ascorbic acid. Ten-eleven translocation 3 (TET3) in oocytes was knocked down by siRNA injection. After ascorbic acid treatment, reprogramming genes were analyzed by realtime reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, relative 5-methylcytosine and 5-hydroxymethylcytosine content in pronucleus were detected by realtime PCR. Results: Ascorbic acid significantly increased the development of porcine embryos produced by SCNT. After SCNT, transcript levels of reprogramming genes, Pou5f1, Sox2, and Klf were significantly increased in blastocysts. Furthermore, ascorbic acid reduced 5-methylcytosine content in pronuclear embryos compared with the control group. Knock down of TET3 in porcine oocytes significantly prevents the demethylation of somatic cell nucleus after SCNT, even if in the presence of ascorbic acid. Conclusion: Ascorbic acid enhanced the development of porcine SCNT embryos via the increased TET3 mediated demethylation of somatic nucleus.

• Clinical and Experimental Reproductive Medicine
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• 제21권1호
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• pp.69-76
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• 1994

The survival and pregnancy rates were compared between non-frozen embryos and cryopreserved embryos at either pronucleate or 2-4 cell stages using the freezing and thawing techniques being identical in both groups were compared with fresh embryos. 496 embryos were frozen with 1, 2-propanediol and sucrose and 117 2-4 cell stages embryos had been thawed and 79.6 and 66.0% of them respectively were survival. Clinical pregnancy rate was 19.2% for embryos frozen at the pronucleate stage and 19.0% for embryos frozen at the 2-4 cell stages while the pregnancy rate of non-frozen embryos was 21.3%. There were no significant difference in the survival and pregnancy rates of embryos frozen at pronucleate and 2-4 cell stages. The current cumulative pregnancy rate per retrieval in all cycles with frozen zygotes is 35.4 %, consid~ erably higher than observed in single transfers of embryos without cryopreservation(21.3%); predicted pregnancy rate after transfer of all frozen embryos is 43.3 %. It is concluded that firstly, the survival and pregnancy rate of cryopreserved embryos at pronucleate or 2-4 cell stages are very similar to those from their fresh embryos and non-frozen embryos and secondly, cryopreservation substantially enhances pregnancy attainment from in vitro fertilization.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.6-6
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• 2002

This study was conducted to investigate the effect of recipient activation time on the nuclear remodeling, chromatin structure, pronuclear formation and in vitro development of bovine nuclear transfer embryos derived from adult ear skin cells. Somatic cells were transferred to enucleated oocytes after quiescent treatments by serum starvation or culture to confluency. Nuclear transfer embryos were activated with a combination of Ca/sup 2+/-ionophore and cycloheximide at 1, 1.5, 2, 2.5, 3, and 5 h after electrofusion. (omitted)

• 한국환경과학회지
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• 제32권3호
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• pp.173-179
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• 2023

Rats are one of the most widely used animals in biomedical sciences because their metabolism and physiology are comparable to humans. In recent years, gene-targeted models have been developed using various animal species utilizing engineered nucleases such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated gene (Cas). It has recently become possible to efficiently transfect CRISPR/Cas into embryos via electroporation. However, electroporation can damage fertilized eggs; therefore, it is important to determine the optimal embryo culture conditions. A standardized approach for routine and reproducible rat transgenesis will render rat models more accessible for research. We performed experiments to obtain rat embryos with efficient superovulation and synchronization, and to investigate the appropriate medium conditions for pronuclear stage embryos subjected to electroporation stimulation for the introduction of engineered nuclease.

• 한국수정란이식학회지
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• 제22권1호
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• pp.53-61
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• 2007

본 연구는 PI한 한국흑염소 수정란의 이식 결과를 통해 수란 흑염소의 수태율에 영향을 미칠 것으로 생각되는 여러 가지 요인을 분석함으로써, 높은 수태율을 얻을 수 있는 수란 흑염소의 최적 조건을 찾아낼 목적으로 수행하였다. 분석 결과, 수태율에 유의적인 영향을 주는 요인들은 발정형태, 수술 빈도, 이식 부위, 황체의 발육 단계, 수정란의 발육단계, 이식된 수정란의 수 등이었다. 자연 발정이 관찰되어 이식된 흑염소(59.1%, 13/22)들이 $CIDR^(R)$로 발정이 유도된 후 이식된 흑염소(36.8%, 118/321)에서보다 높은 수태율을 나타내었으며(P<0.05), 두 번째 수술 받은 흑염소의 수태율(56.5%, 13/23)이 처음 이식 받은 흑염소(36.5%, 116/318)에 비해 수태율이 높았다(P<0.05). 이식한 부위에 따른 수태율은 좌측 난관에 이식한 흑염소(49.0%, 50/102)가 오른쪽 난관에 이식한 흑염소(35.9%, 46/128)에 비해 수태율이 더 높게 나타났으며(P<0.05), 황체의 발육 단계에 따른 수태율에서는 $CH_1$단계(47.5%, 57/120) 출혈체를 가진 수란 흑염소에서 $CH_3(17.9%,\;7/39)$의 출혈체를 가진 수란 흑염소보다 높은 수태율을 얻었다(P<0.01). 수정란의 발육단계에 따른 차이는 난관 이식의 경우에 1세포기 배가 이식된 수란 흑염소의 수태율(51.6%, 49/95)이 4세포기배를 이식한 경우(24.5%, 12/49)보다 높았다(P<0.01). 수정란의 수는 2개를 이식했을 때(27.%, 37/137)보다 3개를 이식했을 때(47.6%, 50/105) 더 높은 수태율을 얻었다(P<0.01). 수태율에 유의적인 영향이 없는 요인들은 난관 유착이나 자궁 유착, 난소 유착, 자궁각의 크기, 황체의 수, 대형 난포의 존재 유무, 이식의 난이도 등이었다 그러나, 중간 크기의 자궁을 가진 수란 흑염소(38.9%, 122/314)에서 직경 5mm 이하의 작은 자궁을 가진 수란 흑염소(20%, 1/5)나 20mm 이상의 큰 자궁을 가진 수란 흑염소(18.2%, 2/11)보다 수태율이 높은 경향을 보였고, 배란 황체가 있는 같은 쪽 난소에 대형 난포가 존재할 경우(53.3%, 16/30)에 존재하지 않는 경우(37.1%, 104/280)보다 수태율이 높아지는 경향을 보였으며, 이식이 쉽게 이루어진 경우(39.2%, 125/319)에 이식이 어렵거나(27.8%, 5/18) 곤란한 경우(0%, 0/3)에서보다 높은 수태율을 얻을 수 있었다. 따라서, 자궁각의 크기나 대형 난포의 존재 유무, 이식의 난이도 등도 수정란 이식 후의 수태율에 영향을 줄 수 있을 것으로 생각된다. 이상의 결과로 볼 때 PI한 한국 흑염소 수정란의 이식 시 높은 수태율을 얻기 위해서는 수란 흑염소는 자연 발정 온 개체를 이용하고, 난관에 이식하고자하는 경우에는 난소에 $CH_1$ 단계의 출혈체가 존재하는 쪽 난관에 1세포기의 수정란을, 그리고 자궁에 이식하는 경우에는 난소에 발육 단계가 $CL_3$인 황체가 존재하는 쪽 자궁각에 중기 배반포나 후기 배반포를 이식하는 것이 가장 바람직하다는 결론을 얻었다.

• 한국가축번식학회지
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• 제19권2호
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• pp.89-93
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• 1995

In this study was demonstrate that retrovirus-mediated gene transfer is one of the promising alternatives to the conventional pronuclear DNA microinjection approach, especially in transferring the exogenous genes into the boving embryos. By co-culturing of zona of zona-free one-cell stage embryos with the retrovirus-producing cells for 24 hours followed by 6 days of culture in virus-free medium, we could get morulae and blastocysts expressing the E. coli LacZ genes which were transferred by our retrovirus vector. The results obtained in this study are summarized as follows : 1. Addition of 5$\mu\textrm{g}$/ml of polybrene in the embryo and virus-producing cell co-culture medium did not affect development of zona-free one-cell embryo. 2. Compared with the intact embryos removal of zona at one-cell stage before co-culturing with the virus-producing cells for one day caused only slight decrease of embryo develpment. 3. Co-culture of 625 zona-free one-cell stage embryos with the virus-producing cells resulted in 65(10.4%) morulae or blastocysts, and 12.3%(8/65) of the morulae or blastocysts were E. coli LacZ positive.

• Clinical and Experimental Reproductive Medicine
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• 제20권1호
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• pp.31-36
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• 1993

The effects of freezing and 1,2-propanediol on early and late pronucleate stage mouse ova were investigated in terms of survival after thawing and development in vitro. The samples were divided into two groups according to different age in pronucleate ova: ova in(1) early pronuclear stage with two distant pronuclei at 18h after hCG injection, and (2) late pronuclear stage with adjacent pronuclei at 30h. Zygotes in the late pronuclear stage have been proven to be more resistant to 1,2-propanediol, showing a significantly higher developmental rate than zygotes in early stage (80.3 versus 66.3%, <0.05), but survival rate was similar in the two groups (91.0 versus 93.5%). After freezing and thawing, survival and developmental rates were decreased in both groups when compared to the control group (54.3 versus 92.3%, 47.7 versus 73.3%. respectively). And developmental rate in the late pronuclear stage zygotes showed significantly higher than in early (55.4 versus 40.0%) after thawing. In conclusion, early pronucleate mouse ova have a lower developmental capacity in vitro and a lower survival rate after freezing and thawing than late ova. These findings suggest that the timing of freezing could be important for survival and further development in vitro in cryopreservation of human pronucleate ova.