• 한국동물생명공학회지
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• 제34권3호
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• pp.148-156
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• 2019

The Transgenic livestock can be useful for the production of disease-resistant animals, pigs for xenotranplantation, animal bioreactor for therapeutic recombinant proteins and disease model animals. Previously, conventional methods without using artificial nuclease-dependent DNA cleavage system were used to produce such transgenic livestock, but their efficiency is known to be low. In the last decade, the development of artificial nucleases such as zinc-finger necleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas has led to more efficient production of knock-out and knock-in transgenic livestock. However, production of knock-in livestock is poor. In mouse, genetically modified mice are produced by coinjecting a pair of knock-in vector, which is a donor DNA, with a artificial nuclease in a pronuclear fertilized egg, but not in livestock. Gene targeting efficiency has been increased with the use of artificial nucleases, but the knock-in efficiency is still low in livestock. In many research now, somatic cell nuclear transfer (SCNT) methods used after selection of cell transfected with artificial nuclease for production of transgenic livestock. In particular, it is necessary to develop a system capable of producing transgenic livestock more efficiently by co-injection of artificial nuclease and knock-in vectors into fertilized eggs.

• 한국가금학회:학술대회논문집
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• 한국가금학회 2004년도 제21차 정기총회 및 학술발표회
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• pp.9-10
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• 2004

형질전환 가금의 생산은 생체반응기(Bioreactor)가금에 의한 고부가가치의 생의약 물질을 저비용, 고효율로 생산할 수 있으며, 배 발달과정 및 유전자 조절기작 규명을 통한 학문적 이용성 등 다양한 분야에 응용될 수 있다. 형질전환 가금을 생산하기 위한 방법 중 닭의 배 발생 초기에 발생하는 성세포(정자 혹은 난자)의 전구세포인 원시생식세포를 이용한 연구가 활발하게 진행되고 있다. 그러나 이를 검증할 원시생식세포 특이적 마커의 부재로 많은 어려움을 겪고 있다. 따라서 본 연구는 원시생식세포의 특성 분석을 위해 PAS(Periodic acid-Schiff) 염색 및 특이항체 (SSEA-1,3,4 & Integrins $\alpha$6, $\beta$l) 그리고 lectins (STA, DBA, ConA, WGA)를 이용하였다. 이번 연구결과를 통한 닭 원시생식세포의 특이적 마커의 개발은 원시생식세포를 이용한 가금의 형질전환 연구에 기여할 것이다.

• Asian-Australasian Journal of Animal Sciences
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• 제30권8호
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• pp.1175-1182
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• 2017

Objective: To create genetically modified goat as a biopharming source of recombinant human lacotoferrin (hLF) with transcription activator-like effector nucleases. Methods: TALENs and targeting vector were transferred into cultured fibroblasts to insert hLF cDNA in the goat beta-lactoglobulin (BLG) locus with homology-directed repair. The gene targeted efficiency was checked using sequencing and TE7I assay. The bi-allelic gene targeted colonies were isolated and confirmed with polymerase chain reaction, and used as donor cells for somatic cell nuclear transfer (SCNT). Results: The targeted efficiency for BLG gene was approximately 10%. Among 12 Bi-allelic gene targeted colonies, five were used in first round SCNT and 4 recipients (23%) were confirmed pregnant at 30 d. In second round SCNT, 7 (53%), 4 (31%), and 3 (23%) recipients were confirmed to be pregnant by ultrasound on 30 d, 60 d, and 90 d. Conclusion: This finding signifies the combined use of TALENs and SCNT can generate biallelic knock-in fibroblasts that can be cloned in a fetus. Therefore, it might lay the foundation for transgenic hLF goat generation and possible use of their mammary gland as a bioreactor for large-scale production of recombinant hLF.

• Journal of Animal Science and Technology
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• 제61권2호
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• pp.61-68
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• 2019

The hG-CSF (human Granulocyte Colony-Stimulating Factor) is a growth and stimulation factor capable of inducing the proliferation of bone marrow cells, several types of leukocytes, among other hematopoietic tissue cells. hG-CSF is used in used to treat anomalies that reder a small number of circulating white blood cells, which may compromise the immune defenses of the affected person. For these reasons, the production of hG-CSF in a bioreactor system using the mammary gland of genetic modified animals is a possibility of adding value to the bovine genetic material and reducing the costs of hG-CSF production in pharmaceutical industry. In this study, we aimed the production of transgenic hG-CSF bovine through the lipofection of bovine primary fibroblasts with an hG-CSF expression cassette and cloning these fibroblasts by the somatic cell nuclear transfer (SCNT) technique. The bovine fibroblasts transfected with the hG-CSF cassette presented a stable insertion of this construct into their genome and were efficiently synchronized to G0/G1 cell cycle stage. The transgenic fibroblasts were cloned by SCNT and produced 103 transferred embryos and 2 pregnancies, one of which reached 7 months of gestation.

• Journal of Plant Biotechnology
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• 제42권1호
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• pp.34-42
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• 2015

본 연구는 약용식물인 적하수오의 부정근과 생리활성물질 대량생산을 위해 생물반응기 배양 조건을 확립하고자 실시되었다. 이를 위하여 생물반응기 배양 시 배지 내 무기물 함량과, auxin의 종류와 농도, sucrose 농도가 적하수오 부정근의 생장과 총 phenolics와 flavonoids 함량에 미치는 영향을 조사하였으며, 배양 중 methyl jasmonate (MeJA)와 salicylic acid (SA)의 첨가가 생리활성물질 축적에 미치는 영향을 조사하였다. 그 결과 $9.84{\mu}M$ IBA와 50 g/L sucrose가 첨가된 1배 MS배지에서 최적의 부정근 생장이 이루어졌으며 생리활성물질 축적도 가장 높았다. MeJA와 SA 처리시 적하수오 부정근의 생장과 생리활성물질 축적은 오히려 감소하였다. 본 연구 결과는 산업적 목적을 위한 적하수오의 부정근과 생리활성물질 대량생산시 기초자료로 활용될 수 있을 것으로 기대된다.

• Reproductive and Developmental Biology
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• 제37권3호
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• pp.91-96
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• 2013

Transgenic chickens have been spotlighted as an highly potent bioreactor for their fecundity, short generation time, and eggs associated with mass production of protein. In this study, we generated transgenic chickens exhibiting oviduct specific expression of human growth hormone fused to human transferrin for oral administration. Gene of the modified growth hormone located at downstream ovalbumin promoter (~3.6 kb) was introduced to stage X blastodermal cell employing retrovirus vector system. Several transgenic chickens were successfully generated. However, genomic analyses showed unexpected deletion within the transgene. The modification of the transgene seemed to occur during germ cell formation because the deletion was detected only from the sperm DNA of the G0 founder animal. There was no evidence of deletion in the somatic cell DNA samples of the same chicken. Consequently, same pattern of the deletion was confirmed in both somatic and germ cells of the G1 progeny.

• 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.

• 한국가축번식학회지
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• 제26권2호
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• pp.95-104
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• 2002

본 연구는 사람의 조혈촉진 유전자(hEPO)가 도입된 형질전환 돼지를 생산하기 위해 사계절동안 수행하였다. 약 8∼15개월령의 순종의 랜드레이스 경산돈 및 미경산돈 42두는 유전자 미세주입을 위한 1세포기 단계의 수정란 채란 및 이식을 위해 사용하였으며, 발정동기화 및 과배란 방법은 PG 600 주입 후 9일간 매일 20mg의 altrenogest를 사료에 첨가하여 급여하였다. Altrenogest를 9일간 급여 후 1,500IU의 PMSG와 500IU의 hCG를 주입하므로서 과배란을 유도하였다. 미세주입을 위한 유전자는 mouse whey acidic protein(mWAP) 프로모터에 hEPO 유전자를 연결하여 준비하였으며, 호르몬 처리후 23두의 공란돈으로 부터 650개의 난자를 회수하였으며, 이 중 83.1%(540/650)는 DNA 미세주입을 위해 전핵을 관찰할 수 있는 1-세포기의 수정란이었다. 이중 유전자가 미세주입 된 543개의 난자를 19두의 수란돈에 이식하였으며 7두의 임신돈으로부터 47두의 자돈을 생산하였다. 생산된 자돈 47두로부터 꼬리조직으로부터 분리된 DNA의 PCR 검정 결과 수컷 1두가 형질전환 양성반응을 나타내어 2.13%의 형질전환율을 나타내었으며, 이러한 연구의 결과는 생체반응기(bioreactor)연구에 있어서 형질전환 돼지생산의 성공적이며 유용한 정보를 제공할 것으로 사료된다.

• Journal of Microbiology and Biotechnology
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• 제32권5호
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• pp.630-637
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• 2022

The objective of this study was to optimize industrial-grade media for improving the biomass production of Weissella cibaria JW15 (JW15) using a statistical approach. Eleven variables comprising three carbon sources (glucose, fructose, and sucrose), three nitrogen sources (protease peptone, yeast extract, and soy peptone), and five mineral sources (K₂HPO₄, potassium citrate, ⳑ-cysteine phosphate, MgSO₄, and MnSO₄) were screened by using the Plackett-Burman design. Consequently, glucose, sucrose, and soy peptone were used as significant variables in response surface methodology (RSM). The composition of the optimal medium (OM) was 22.35 g/l glucose, 15.57 g/l sucrose, and 10.05 g/l soy peptone, 2.0 g/l K₂HPO₄, 5.0 g/l sodium acetate, 0.1 g/l MgSO₄·7H₂O, 0.05 g/l MnSO₄·H₂O, and 1.0 g/l Tween 80. The OM significantly improved the biomass production of JW15 over an established commercial medium (MRS). After fermenting OM, the dry cell weight of JW15 was 4.89 g/l, which was comparable to the predicted value (4.77 g/l), and 1.67 times higher than that of the MRS medium (3.02 g/l). Correspondingly, JW15 showed a rapid and increased production of lactic and acetic acid in the OM. To perform a scale-up validation, batch fermentation was executed in a 5-l bioreactor at 37℃ with or without a pH control at 6.0 ± 0.1. The biomass production of JW15 significantly improved (1.98 times higher) under the pH control, and the cost of OM was reduced by two-thirds compared to that in the MRS medium. In conclusion, OM may be utilized for mass producing JW15 for industrial use.

• 한국수정란이식학회지
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• 제32권3호
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• pp.227-233
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• 2017

Pig has been known to be one of the most feasible animals as a bioreactor to produce pharmaceuticals in milk and as a mediator in xenotransplantation research. Previously, we generated transgenic pigs for both purposes, which were expressing Factor 8, vWF, hTPA, and hEPO in milk, along with expression of MCP at GalT gene locus ($GalT^{-MCP/-MCP}$) as well as expressing MCP at GalT gene loci with CD73 expression ($GalT^{-MCP/+}/CD73$). In this study, we performed comparative analyses of sperm parameters between wild type male (WT) pig and those transgenic males to examine the effects of transgenes integrated into the pigs on motility, morphology, viability, and acrosome integrity of the spermatozoa. Our results showed that the rates of actively motile spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 85.0%, 83.3%, 82.5%, 83.3%, 82.5%, 77.5%, and 78.7%, respectively. Whereas, the rates of morphologically normal spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 90.0%, 80.0%, 80.0%, 83.3%, 85.0%, 91.8%, and 80.8%, respectively. In addition, the viability in spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 93.9%, 82.4%, 89.9%, 83.9%, 87.4%, 92.8%, and 83.6%, respectively. The rates of spermatozoa with normal acrosome integrity in WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 98.1%, 98.6%, 98.6%, 98.7%, 98.1%, 99.5%, and 95.1%, respectively. There were no significant differences in motility, morphology, viability, and acrosome integrity of the spermatozoa among WT, Factor 8, vWF, hTPA, and hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs. These mean that neither random integration nor targeted integration of the transgene into chromosome of pig effect on characteristics of spermatozoa. Ultimately, the transgenic male pigs subjected in this study could apply to propagate their progenies for production of human therapeutic proteins and advancing the xenotransplantation research.