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Joohyun Shim,Nayoung Ko,Yongjin Lee,Hyoung-joo Kim,Jae-kyung Park,Kimyung Choi 한국동물생명공학회(구 한국동물번식학회) 2017 Reproductive & Developmental Biology(Supplement) Vol.41 No.2
Genetically modified pigs have been considered valuable models of human disease and donors for xenotransplantation. Here, we used Zinc finger nucleases (ZFNs) to knock out the Yucatan miniature pig α-1,3-galactosyltransferase (GGTA1) gene, which generates Gal epitopes that trigger hyperacute immune rejection in pig-to-human transplantation. ZFNs were designed to cleave a region of the GGTA1 gene. Biallelic GTKO cell lines were established from single cell colonies of ear fibroblasts derived from Yucatan miniature pigs following transfection by electroporation. Two cell lines were selected as donor cell line for somatic cell nuclear transfer (SCNT) for the generation of GTKO pigs. The reconstructed GTKO embryos were subsequently transferred into two recipient gilts, of which one became pregnant. We obtained four live piglets and one stillborn. Genotyping of all cloned individuals was performed. The Gal expression in the fibroblasts of all piglets was analyzed by fluorescence activated cell sorting (FACS) and western blotting. Sequencing analyses of the target site confirmed the homozygous GGTA1-null mutation in all fetuses and piglets, consistent with the genotype of the donor cells. Furthermore, FACS and western blotting analyses demonstrated that Gal epitopes were completely absent from the fibroblasts of all GTKO piglets.
심주현(Joohyun Shim),김헌정(Heonjeong Kim),오세윤(Seyun Oh),김동수(Dongsoo Kim),이철수(Cheolsoo Lee),김승환(Franz Kim) (사)한국CDE학회 2012 한국 CAD/CAM 학회 학술발표회 논문집 Vol.2012 No.2
기존 블록조립 정반 배치 시스템은 화면상에 정반의 레이아웃을 사각형상으로 표현하고, 배치되는 블록을 대표 형상(사각, 삼각, 사다리꼴 등)만으로 표현하여 배치를 수행하였다. 이러한 배치 작업은 공정 담당자의 경험과 직관이 매우 중요하며, 작업자의 숙련도에 따라 배치 작업의 신뢰도에 큰 영향을 주었다. 또한 모든 배치 작업이 수작업으로 이루어져 공정계획의 효율성과 신뢰성을 보장하지 못하는 문제점을 안고 있었으며, 블록의 크기 정보만으로 블록을 표현하기 때문에 실제 조립 현장의 현황 및 간섭을 예측하지 못하였다. 본 논문에서는 이러한 문제점을 해결하고자 CAD System(AM)의 모델 데이터와 Assembly Planning 정보를 활용하여 3D 블록 형상을 재현하고, 2D 경계 곡선을 추출하여 실 배치에 적용하였으며, 공장 레이아웃 Modeling Tool(Factory CAD)로 대조립 공장을 형상화하여 실제 정반의 정확한 위치 정보를 사용할 수 있도록 구현하였다. 또한, 수작업에 의존하던 배치 작업을 자동화하여 배치 효율성을 높였으며, 다양한 제약조건을 적용하여 일정 지연을 최소화하는 최적 배치 알고리즘을 구현하였다. 제안된 최적배치 알고리즘은 블록 선택 단계, 작업반 선택 단계 그리고 작업반 내 배치 위치 판단 단계의 3 단계로 구현되었으며, 배치 대상 블록의 일정정보, 중량, 예산 및 배치 순서 제약 속성과 통로, Crane 의 이동 방향 및 블록의 크기를 고려하여 배치 위치와 일정을 결정하였다. 최적배치 알고리즘 적용으로 인해 기존 수작업 System 의 단점을 보완하고 효율성과 신뢰성을 높이는데 기여하였다.
Choi, Kimyung,Shim, Joohyun,Ko, Nayoung,Park, Joonghoon Asian Australasian Association of Animal Productio 2020 Animal Bioscience Vol.33 No.2
Objective: Specific genomic sites can be recognized and permanently modified by genome editing. The discovery of endonucleases has advanced genome editing in pigs, attenuating xenograft rejection and cross-species disease transmission. However, off-target mutagenesis caused by these nucleases is a major barrier to putative clinical applications. Furthermore, off-target mutagenesis by genome editing has not yet been addressed in pigs. Methods: Here, we generated genetically inheritable α-1,3-galactosyltransferase (GGTA1) knockout Yucatan miniature pigs by combining transcription activator-like effector nuclease (TALEN) and nuclear transfer. For precise estimation of genomic mutations induced by TALEN in GGTA1 knockout pigs, we obtained the whole-genome sequence of the donor cells for use as an internal control genome. Results: In-depth whole-genome sequencing analysis demonstrated that TALEN-mediated GGTA1 knockout pigs had a comparable mutation rate to homologous recombination-treated pigs and wild-type strain controls. RNA sequencing analysis associated with genomic mutations revealed that TALEN-induced off-target mutations had no discernable effect on RNA transcript abundance. Conclusion: Therefore, TALEN appears to be a precise and safe tool for generating genomeedited pigs, and the TALEN-mediated GGTA1 knockout Yucatan miniature pigs produced in this study can serve as a safe and effective organ and tissue resource for clinical applications.
Yongjin Lee,Joohyun Shim,Nayoung Ko,Hyoung-Joo Kim,Jeakyoung Park,Kwak Kyungmin,Kimyung Choi 한국동물생명공학회(구 한국동물번식학회) 2018 발생공학 국제심포지엄 및 학술대회 Vol.2018 No.06
Alanine known as non-essential amino acid was detected at high concentration in reproductive tracts and follicular fluid in developing porcine antral follicle. The purpose of this study was to determine the effect of alanine supplementation during in vitro maturation (IVM) of porcine oocyte. We investigated nuclear maturation, intraoocyte glutathione (GSH) contents in metaphase II (MII) oocytes, and subsequent embryonic development. And also, We detected the gene expression pattern in MII oocytes, early embryo and blastocyst derived parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). The base medium for IVM was North Carolina State University-23 (NCSU-23) medium, modified by supplementing 10 ng/mL EGF, 0.5 ug/mL FSH, and 0.5 ug/ LH, replacing BSA with 0.1% (w/v) PVA, and deleting glutamine. Alanine of various concentrations (0, 0.363, 1, 5, and 10 mM) were added to base IVM medium. The proportion of mature oocyte after IVM did not increase by alanine treatment at various concentrations. However, The intraoocyte GSH content was higher (p<0.05) in oocytes treated with 0.363 mM alanine (1.17±0.01 pixels per oocyte) than non-treated oocytes (1.00±0.02 pixels per oocyte). Blastocyst formation of PA (31.2±2.5% vs. 19.8±2.5%) and SCNT (20.5±1.9% vs. 10.1±2.0%) embryos was significantly (p<0.05) improved by treatment with 0.363 mM alanine during IVM compared with embryos derived from the non-treated oocytes. Supplementation of oocytes IVM medium with 0.363 mM alanine significantly (p<0.05) increased the gene expression of POU5F1 and FGFR2 levels in early embryo and blastocyst derived PA and SCNT embryos. In MII oocytes, transcript levels of POU5F1 and FGFR2 as well as CDK1 gene were significantly (p<0.05) increased in 0.363 mM alanine-treated oocytes. Our results demonstrate that treatment with 0.363 mM alanine during pig oocyte maturation improves developmental competence after PA and SCNT by influencing cytoplasmic maturation, such as improved GSH content in IVM oocyte and increasing gene expression associated with embryonic development in oocyte and embryo.
Hyoung-Joo Kim,Joohyun Shim,Nayoung Ko,Yongjin Lee,Jae-Kyung Park,Kyungmin Kwak,Jun-Hyeong Kim,Pulip Kang,Jeong-Woong Lee,Hyunil Kim,Kimyung Choi 한국실험동물학회 2021 한국실험동물학회 학술발표대회 논문집 Vol.2021 No.7
The COVID-19 pandemic is caused by SARS-CoV-2, which continues to raise, public health concerns worldwide. Coronaviruses have an outer crown-like spike protein that binds to the human Angiotensin-converting enzyme 2 (ACE2) gene, resulting in infection via endocytosis. Therefore, research on hACE2 is expected to be critical for developing our understaining of COVID-19. To facilitate this, we developed Yucatan miniature pigs expressing hACE2 as animal models for COVID-19 research. First, vector containing hACE2 gene, FLAG tag, and GFP was constructed using the CMV promoter. Three lines of stable cell lines with hACE2 protein expression were created by transfecting Yucatan miniature pig ear fibroblasts with the constructed vector. The established cells then underwent somatic cell nuclear transfer, and were transferred to surrogate sows as donor cells, resulting in the successful production of four transgenic cloned pigs. PCR analysis confirmed that the hACE2 gene was inserted into the genome, and that hACE2 mRNA was well-expressed in the lung, heart, and small intestine. In addition, differences in protein expression between transgenic clones and wild-type pigs were confirmed using ear fibroblasts. Finally, karyotyping and fluorescence in situ hybridization analysis revealed that the transgenic cloned pigs had the same number of chromosomes as normal pigs (36 chromosomes, plus two sex chromosomes), and that the hACE2 gene was inserted into chromosome 17. In this study, we succesfully produced transgenic Yucatan miniature pigs expressing the hACE2 gene for use as prelinical COVID-19 target models. *This work has supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2020M3A9I2105803).
Generation of the GGTA1/CMAH/hCD46 Genetically Modified Pigs for Xenotransplantation
Hyoung-Joo Kim,Joohyun Shim,Nayoung Ko,Yongjin Lee,Jae-Kyung Park,Kimyung Choi 한국동물생명공학회(구 한국동물번식학회) 2017 Reproductive & Developmental Biology(Supplement) Vol.41 No.2
The demand for organ transplantation has rapidly increased all over the world during the past decade. Genetically modified pigs provide a solution to the severe shortage of organs available for human transplantation. Porcine α-1,3-galactosyltransferase (GGTA1) gene is generates Gal-T epitopes that trigger hyperacute rejection in pig-to-human transplantation. Since production of GGTA1 knock-out pigs in 2002, non-gal antigens are considered to be the next xenoantigen involved in the rejection phenomenon. Here, we targeted the GGTA1 and CMP-Neu5Ac hydroxylase (CMAH) genes with CRISPR-Cas9 systems resulting in double knock-out pigs that no longer express α-Gal or Neu5Gc. Similar to GGTA1 gene, CMAH is widely expressed on the endothelial cells of many mammals except humans and this epitope is a potential porcine target for the antinon- gal antibody in humans. CMAH is responsible for the expression of Neu5Gc that key non-gal antigen. Additionally, hCD46 controls complement activation and when this gene expressed sufficiently as a transgene protects xenografts against complement-mediated rejection. This is report to describe generation of transgenic pigs that modify GGTA1, CMAH and hCD46. We expect to remove α-gal and Neu5Gc antigens and express hCD46 from pig for reducing human antibody mediated cytotoxicity.