• 한국수정란이식학회지
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• 제23권2호
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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.

• 한국가금학회지
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• 제27권2호
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• pp.155-161
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• 2000

Unfortunately, there is no technique which is stable and repetitive to produce transgenic chicken, although various ways of gene transfer including PGC-and embryonic cell-mediated gene transfer, DNA microinjection, virus inoculation and sperm cells have been employed. The aims of this study were 세 develop and establish such a stable, repetitive and efficient way of gene transfer giving a faithful gene expression during development after the reconstruction of embryo in an UV-irradiated egg. A dual reporter plasmid (pJJ9), a fusion gene containing lacZ and GFP driven by a CMV promoter was used to exploit either merits of both reporting markers. lacZ with strong signal or GFP with vital marking. Electroporated embryonic blastodermal cells (EBCs) in the presence of the pJJ9 DNA faithfully showed 377 bp PCR product and lacZ or GFP expressions in the identical cells in vitro of in vivo. Furthermore, analyses of expression pattern of the foreign DNA demonstrated that microinjected EBCs cells into the UV-irradiated recipient egg should participate in normal developmental process, for example, proliferation and differentiation into various tissues. Thirty percentages of the manipulated eggs showed lacZ expression in their tissues. These results together with the specific procedures used in this study should facilitate avian transgenesis.

• 한국수정란이식학회지
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• 제33권4호
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• pp.245-254
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• 2018

Pigs are considered as optimal donor animal for the successful xenotransplantation. To increase the possibility of clinical application, genetic modification to increase compatibility with human is an important and essential process. Genetic modification technique has been developed and improved to produce genetically modified pigs rapidly. CRISPR/Cas9 system is widely used in various fields including the production of transgenic animals and also can be enable multiple gene modifications. In this study, we developed new gene targeting vector and enrichment system for the rapid and efficient selection of genetically modified cells. We conducted co-transfection with two targeting vectors for simultaneous inactivation of two genes and enrichment of the genetically modified cells using MACS. After this efficient enrichment, genotypic analysis of each colony showed that colonies which have genetic modifications on both genes were confirmed with high efficiency. Somatic cell nuclear transfer was conducted with established donor cells and genetically modified pigs were successfully produced. Genotypic and phenotypic analysis of generated pigs showed identical genotypes with donor cells and no surface expression of ${\alpha}$-Gal and HD antigens. Furthermore, functional analysis using pooled human serum revealed dramatically reduction of human natural antibody (IgG and IgM) binding level and natural antibody-mediated cytotoxicity. In conclusion, the constructed vector and enrichment system using MACS used in this study is efficient and useful to generate genetically modified donor cells with multiple genetic alterations and lead to an efficient production of genetically modified pigs.

• 한국발생생물학회지:발생과생식
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• 제13권4호
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• pp.305-312
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• 2009

단분화성 정원줄기세포의 장기간 체외배양 중에 확립되는 다분화능 정원줄기세포는 배아줄기세포와 유사한 특성을 가져 3배엽성 세포로 체외분화가 가능하며 기형종을 형성할 수 있다. 본 연구에서는 선행 연구를 통해 outbred 생쥐(ICR strain)로부터 확립된 다분화능 정원줄기세포의 형질전환 가능성을 확인하며, 배아 내로 주입하여 유전적 키메라를 형성하는 효율을 배아줄기세포와의 비교를 통하여 검증하고자 하였다. 다분화능 정원줄기세포를 넣은 배아로부터 태어난 산자는 총 47마리(4.8%)가 태어나, 67마리(11.7%)가 태어난 배아줄기세포군에 비해 그 효율이 낮았다(P<0.05). 그러나 산자들 중의 키메라 생쥐의 비율은 다분화능 정원줄기세포 군으로 부터 3마리(6.4%)가 태어나 배아줄기세포 군으로부터 태어난 5마리(7.5%)와 유사하였다(P>0.05). 태어난 유전자변형 생쥐의 장기를 확인한 결과, 췌장, 심장, 뇌, 근육, 위, 피부, 정소에 GFP가 발현되는 것을 확인하였다. 또한 배아의 근육, 위, 뼈 등에서 anti-GFP 항체의 발현을 확인하였다. 이상의 결과를 종합하면 outbred 생쥐로부터 확립된 다분화능 정원줄기세포가 inbred 생쥐로부터 확립된 배아줄기세포와 마찬가지로 키메라 생쥐를 생산할 수 있는 전분화능을 가짐을 확인하였고, 새로운 유전자변형 동물의 생산을 위한 매개체로서의 가능성을 가진 것으로 여겨진다.

• Journal of Plant Biotechnology
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• 제37권4호
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• pp.511-516
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• 2010

아그로박테리움 매개에 의한 형질전환 기술을 이용하여 국내에서 육성된 품종 'Sweet Yellow'로부터 유도된 체세포배 (배발생캘러스 포함)로 intron-GUS유전자가 전이된 식물체를 획득하기까지의 과정이 제시되었다. Intron-GUS 유전자를 포함하고 있는 Agrobacterium tumefaciens AgL1(O.D=0.7~1.6)에 30분 감염시켜 3일간 공동배양 한 후 $4^{\circ}C$에서 7일간의 저온처리를 거친 후 cefotaxim $250\;mg{\cdot}L^{-1}$ 첨가 체세포배발아 배지에 배양된 체세포배 (배발생캘러스 포함)들 대부분으로 유전자가 전이된 것을 GUS transient assay에 의해 확인하였다. Intron-GUS유전자가 전이된 체세포배 (배발생캘러스 포함)로부터 신초원기를 유도한 후 신초를 재분화시켰고, 재분화된 신초로부터 다신초가 형성되도록 하였다. 다신초로부터 신초의 일부를 떼어 GUS transient assay 분석을 실시하여 intron-GUS 유전자의 발현을 확인한 후 발근시켜 순화 후 온실로 옮겼다. GUS transient assay에 의해 확인된 유전자 발현율은 100%였다.

• 한국가축번식학회지
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• 제25권4호
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• pp.305-315
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• 2001

본 연구의 목적은 외래 EGFP 유전자를 분화이전의 웅성생식세포에 도입한 후 이를 난모세포내에 미세주입하여 형질전환동물을 생산하는 기술을 개발하는 데에 있다. 이를 위하여 반수체 정자세포에서 특이적으로 발현하는 생쥐의 mTP1과 햄스터의 hPrm2 유전자 발현 시기를 RT-PCR로 조사한 결과 그시기는 생쥐와 햄스터에서 각각 18일령과 20일령으로 확인되었다. 이에 따라 외래 유전자의 침입이 용이한 감수분열 직전단계인 17일령의 생쥐와 19일령의 햄스터 정자세포를 EGFP 유전자가 포함된 배양액에 부유시킨 다음, 전기자극을 부여한 결과 0.18 ㎸/cm의 전기자극을 가한 후 72시간 배양한 정자세포의 28.5%와 32.1%에서 EGFP 유전자가 발현되는 것으로 확인되었다. EGFP유전자가 도입된 반수체 정자의 수정 및 발달 능력을 검증하기 위하여, 이들 정자세포를 햄스터 난자 내에 미세주입 하였으나, 형광현미경하에서는 EGFP유전자의 발현은 관찰할 수 없었다. 이에 이들 난자를 공시하여 PCR분석을 실시한 결과, 약 44%의 수정란에서 EGFP 유전자의 존재가 확인되었다. 이러한 결과로 보아 반수체 정자세포는 외래 유전자를 난자 내에 도입하기 위한 운반체로 이용될 수 있을 것으로 생각된다.

• 한국자원식물학회지
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• 제10권2호
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• pp.107-113
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• 1997

화이트 클로버의 배축, 잎, 미숙배 유래의 embryogenic callus에 식물 binary vector인 pBI121을 포함하는 A. tumefaciens LBA4404를 접종하여 효과적으로 화이트 클로버를 형질전환시켰다. A. tumefaciens를 이용한 화이트 클로버의 형질전환은 acetosyringone을 사용함으로써 품종간의 차이가 없이 배발생 캘러스에서 16-19%를 보였다. 재분화 식물체의 PCR 및 Northern 분서글 통하여 형질 전환된 화이트 클로버의 염색체내에 GUS 유전자가 안정되게 도입되었고 식물체내에서 mRAN로 발현됨을 확인하였다. 또한, GUS 유전자가 식물체내에서 단백질로 발현됨을 확인하기 위하여 형질 전환되어진 화이트 클로버부터 단백질을 추출하고 분광분석법에 의하여 GUS의 활성을 측정하였으며, 시료간에 약간의 차이는 있으나 유의적인 GUS 활성을 확인하였다.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2017년도 9th Asian Crop Science Association conference
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• pp.115-115
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• 2017

Zinc finger proteins constitute a large family which has been studied to have various functions in different organisms. Tandem CCCH zinc finger proteins (TZFs), members of the zinc finger protein family, are known to participate as post-transcriptional regulators of gene expression in eukaryotes. Here, we showed that the OsZn15, a gene for tandem CCCH zinc finger protein, is induced by abiotic stress and its overexpression in transgenic rice plants (PGD1:OsZn15) gains higher drought tolerance. Gene expression analysis of promoter:GFP plants revealed that OsZn15 is specifically expressed in anther and embryo, but not in vegetative organs. In-field evaluation, grain yield was higher in the PGD1:OsZn15 than nontransgenic plants under drought conditions. Interestingly, OsZn15 is shown to not only localize at nucleus but also co-localize with both processing bodies (PB) and stress granules (SG), two messenger ribo-nucleoprotein complexes which are known to activate by forming cytoplasmic foci under stress conditions. In sum, these results suggest that OsZn15 increases drought stress tolerance of rice probably by participating in RNA turnover in PB and SG.

• 한국동물생명공학회지
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• 제36권1호
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• pp.17-24
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• 2021

In this study, the potential toxicity of isoprocarb was demonstrated using zebrafish embryos. We treated isoprocarb (0, 29, and 58 mg/L) to the zebrafish embryos for 72 h then, we estimated morphological changes and apoptotic cell numbers. The increasing extent of apoptosis from the anterior to posterior region of developing zebrafish larvae was correlated with toxicity in the overall development process, including growth and normal organ formation. The appearance of abnormalities in the isoprocarb-treated groups in comparison to normal developing zebrafish larvae was verified using quantitative image analysis based on ImageJ software program. The vascular system comprising a complex interconnection of blood vessels was visualized in vessel-fluorescent transgenic zebrafish (fli1:eGFP). The main vasculature was malformed on isoprocarb treatment, and this was also related to cardiac defects. Taken together, normal embryonic development in zebrafish was interrupted owing to the acute toxicity of isoprocarb.

• 한국동물생명공학회지
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• 제37권3호
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• pp.176-182
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• 2022

Norflurazon is widely used on agricultural lands and has a high potential to pollute water sources. However, its effects on fish have not been fully elucidated. The purpose of our study was to determine whether norflurazon adversely affects the developmental stage of zebrafish, which are frequently used as a model system to evaluate the environmental impact of pollutants. Norflurazon interfered with the hatching of zebrafish embryos and induced several sublethal deformities including body length reduction, increased yolk sac volume, and enlargement of the pericardial region. We further examined the cardiotoxicity of norflurazon in the flk1:eGFP transgenic zebrafish line. The vascular network, mainly in the brain region, was significantly disrupted in norflurazon-exposed zebrafish. In addition, due to the failure of cardiac looping, norflurazon-exposed zebrafish had an abnormal cardiac structure. These developmental abnormalities were related to the apoptotic process triggered by norflurazon. Overall, the present study demonstrated the non-target toxicity of norflurazon by analyzing the hazardous effects of norflurazon on developing zebrafish.