공여세포 처리 조건이 형질전환 복제돼지 생산에 미치는 영향

권대진,곽태욱,오건봉,김동훈,양병철,임기순,김진회,박진기,황성수,Kwon, Dae-Jin,Kwak, Tae-Uk,Oh, Keon-Bong,Kim, Dong-Hoon,Yang, Byoung-Chul,Im, Gi-Sun,Kim, Jin-Hoi,Park, Jin-Ki,Hwang, Seong-Soo 한국동물번식학회 2011 Reproductive & developmental biology Vol.35 No.3

This study was conducted to investigate the effects of donor cell treatments on the production of transgenic cloned piglets. Ear fibroblast cell obtained from NIH MHC Inbred minipig was used as control. The GalT knock-out/CD45 knock-in (GalT/CD46) transgenic cell lines were established and used as donor cells. The reconstructed GalT/CD46 embryos were surgically transferred into oviduct of naturally cycling surrogate sows (Landrace ${\times}$ Yorkshire) on the second day of standing estrus. Unlike control (1.2 kV/cm, 75.4%), the fusion rate of the GalT/CDl6 donor cells was significantly higher in 1.5 kV/cm, (84.5%) than that of 1.25 kV/cm, (20.2%) (p<0.01). When the number of the transferred embryos were more than 129, the pregnancy and delivery rates were increased to 13/20 (65%) and 5/20 (25%) compared to less then 100 group [1/6 (16.7%) and 0/6 (0%)], respectively. To analyze the effect of donor cell culture condition on pregnancy and delivery rates, the GalT/CD46 donor cells were cultured with DMEM or serum reduced medium. In serum reduced medium group, the pregnancy and delivery rates were improved to 8/12 (66.7%) and 5/12 (41.7%) compared to DMEM group [3/7 (42.9%) and 0/7 (0%)], respectively. In conclusion, it can be postulated that an appropriate fusion condition and culture system is essential factors to increase the efficiency of the production of transgenic cloned piglets.

항생제 선별 없이 alpha 1,3-Galactosyltransferase 유전자 넉아웃 기반 Membrane Cofactor Protein과 Thrombomodulin 유전자를 동시에 발현하는 세포라인 확립

이해선(Haesun Lee),박미령(Mi-Ryung Park),오건봉(Keon Bong Oh) 한국산학기술학회 2021 한국산학기술학회논문지 Vol.22 No.12

돼지와 영장류의 면역 차이로 이종이식 돼지 장기에서 보체 활성화, 염증반응 그리고 혈액 응고 등의 거부반응이 발생하며 수여 영장류를 사망에 이르게 한다. 인위적으로 Membrane cofactor protein(MCP) 및 Thrombomodulin(TBM)을 발현시키는 것은 이러한 거부반응으로부터 세포를 보호하고 영장류 생존기간을 연장한다고 보고되었으며 이전 연구에서 코돈 최적화 MCP(mMCP) 와 TBM cDNA를 활용한 발현 벡터를 구축한 바 있다. 본 연구의 목적은 항생제 저항성 유전자를 이용하지 않고 형질전환 세포 선별 시스템을 확립하며 alpha-1,3-Galactosyltransferase knock-out(GTKO)/mMCP/TBM 세포 라인을 개발하는 것이다. Nucleofection을 통해 mMCP/TBM 동시 발현 벡터를 도입한 GTKO 세포를 MCP에 대한 항체를 이용하여 MCP를 발현하는 세포만을 두 차례 분리하였다. 이를 통해 분리한 세포는 단일세포가 되도록 배양하였으며 유세포분석을 통한 MCP 발현 수준에 따라 단일 클론의 세포라인을 분류하여 선별하였다. 최종적으로 MCP 단백질 발현이 90% 이상인 GTKO/mMCP/TBM 유전자형 세포 라인 4종을 개발하였다. 선별된 클론들은 GTKO/mMCP/TBM 돼지 생산을 위한 핵이식 공여세포로 활용할 예정이다. 항생제 저항성 유전자를 사용하지 않는 선별 기술은 향후 의료용 원료 동물 생산에 효과적으로 활용될 수 있을 것으로 기대된다. The immunological barriers between pigs and primates result in complement activation, inflammation, and coagulation in xenografts, consequently leading to the recipient"s death. The intentional co-expression of membrane cofactor protein (MCP) and thrombomodulin (TBM) in pigs is known to contribute to the prolonged survival of xenografted recipient primates. In a previous study, we had constructed MCP/TBM co-expression vectors using codon-optimized MCP (mMCP) and TBM cDNA. The objectives of this study were to establish an antibiotic-free selection system and generate a pig cell line carrying mMCP and TBM expression vectors based on the alpha-1,3-galactosyltransferase knock-out (GTKO) genetic background. The mMCP/TBM co-expression vectors were introduced into GTKO pig fibroblasts. We isolated and selected only MCP-expressing cells using anti-MCP antibodies and cell separators twice. Additionally, we performed single-cell separation and clonal culture depending on the MCP expression level. Finally, we were able to obtain four GTKO/mMCP/TBM clones expressing MCP at levels of 90% and over. The selected clones will be used as donor cells for nuclear somatic cell transfer to produce GTKO/mMCP/TBM pigs for xenotransplantation. We believe that the antibiotic-free selection system established in this study could be useful to produce biomedical source animals without the antibiotic resistance gene.

돼지 형질전환 체세포에서 pWAP promoter 영역에서의 DNA 메틸화 비교

박미령(Mi-Ryung Park),이해선(Haesun Lee),오건봉(Keon Bong Oh) 한국산학기술학회 2021 한국산학기술학회논문지 Vol.22 No.12

DNA methylation는 포유동물에 있어 세포 기능 및 유전자의 안정화 조절로 생명을 유지하는 역할을 한다. 본 연구에서는 돼지 귀조직 유래 섬유아 세포에 돼지 유청 단백질과 사람 조직 플라스미노겐 벡터를 도입하여 메틸레이션 영역에 따른 변화를 비교 분석하였다. pWAP promoter 내 메틸레이션 패턴을 분석하기 위하여 동일한 passage 단계에 있는 세포를 배양하여 채취하였으며, bisulfite sequencing 방법으로 분석을 진행하였다. 그 결과 pWAP promoter 내 upstream 영역 두 부분에서 CpG islands가 있음을 확인하였다. pWAP promoter를 도입한 세포의 경우 5 region의 유전자 도입 그룹에서 유의적으로 메틸이션 패턴이 높게 나타남을 확인하였다. 또한, htPA 벡터를 도입한 세포의 경우 htPA1_4 그룹에서 유의적으로 높은 메틸레이션이 확인되었다. 메틸레이션 양상은 CpC islands 영역과 구축된 세포주에 따라 각기 다르게 나타남을 확인하였다. 이러한 결과는 프로모터 선별 및 세포주 구축이 형질전환 동물을 생산하는 데 있어 중요하게 작용할 수 있을 것으로 여겨지며, 유전자의 기능에 대한 연구에도 기초 자료로 활용 될 수 있을 것으로 사료된다. DNA methylation is vital in maintaining genomic stability and modulating cellular functions in mammalian cells. This study was performed to identify the differentially methylated region (DMR) in porcine whey acid protein (pWAP) and the human tissue plasminogen activator (htPA) vector of porcine ear fibroblast cells. To investigate the methylation patterns of the WAP promoter, cells were collected from the same passage and analyzed by the bisulfite sequencing method. Results showed that two cytosine-guanine dinucleotide (CpG) islands are present in the promoter region of pWAP. The methylation level of the CpG island 5 region was significantly higher in the pWAP transfected somatic cells than in the wild type (WT) somatic cells. In the htPA, transgenic cells were significantly higher in htPA1_4 cells than the other htPA transfected cells. The changing methylation pattern was observed in the promoter regions and established transgenic cell lines. Our data indicate that promoter selection and cell line construction can play an important role in the production of transgenic animals. Thus, these results can be used as basic data for research on the function of genes.

임신말기 자궁내 발육상태와 자궁적출 시기가 특정질병 제어 형질전환 복제동물 생후 생존에 미치는 영향

우제석(Jae-Seok Woo),황성수(Seongsoo Hwang),오건봉(Keon Bong Oh),이휘철(Hwi-Cheul Lee),양병철(Byoung-Chul Yang),임기순(Gi-Sun Im),이명식(Myeung Sik Lee),김민규(Minkyu Kim),노환국(Whangook Nho),박수봉(Soo-Bong Park),홍성구(Sunggoo Hon 충남대학교 농업과학연구소 2011 농업과학연구 Vol.38 No.4

Bioorgan transgenic cloned mini pig has a problem of growth retardation in uterus during end of pregnancy so that survival rate is very low in newborn piglet. In order to support their life after birth, cesarean section of fetus with sufficient growth in uterus was tested in this study. First of all, fetus growth measured using a ultrasound scanner during pregnancy in transgenic mini pig, comparing normal pig. After 113 days for delivering, fetus was removed out of uterus. Fetus growth for normal pig was 1.8 cm at 4weeks and 14.4 cm at end of pregnancy (15 weeks). At 113 days, fetus growth was 15.9±4 cm in ultrasound scanner and real growth measurement from fetus removal out of uterus was 16.0±2 cm. It is very a similar result between measurement of ultrasound scanner and real measurement. Therefore, using ultrasound scanner for measuring fetus growth will be useful to predict fetus growth in uterus.

저산소 상태에서 우르솔산이 배아줄기세포 성장에 미치는 효과

한기연(Gi Yeon Han),박재홍(Jae Hong Park),오건봉(Keon Bong Oh),이세중(Sei-Jung Lee) 한국생명과학회 2013 생명과학회지 Vol.23 No.10

우르솔산(Ursolic acid)은 다양한 약재, 과일 그리고 야채 등으로 부터 분리되는 식물성 생리활성물질로서, 천연항산화제로 널리 알려져 있지만, 배아줄기세포에서 우르솔산의 기능에 대한 연구는 아직까지 잘 이해되지 않고 있다. 본 연구에서는 저산소 상태에서 우르솔산이 배아줄기세포의 성장에 미치는 효과에 대해 조사하였다. 48시간 동안의 저산소 환경은 배아줄기세포의 미분화상태 및 전능성을 유지에 있어서 영향을 미치지 않았지만, 세포 내활성산소종의 지속적인 생산과 이로 인한 lactate dehydrogenase 활성을 촉진 시켰다. 저산소에 의해 유도된 배아줄기세포의 산화적 스트레스는 30 μM의 우르솔산의 처리로 인해 유의적으로 감소되었으며, 이러한 우르솔산의 활성산소 소거능력은 항산화제인 N-acetyl-cysteine (NAC)의 효과와 유사하였다. 저산소 환경은 또한 유의적으로 배아줄기세포의 생존과 증식을 감소 시킬 뿐만 아니라, 세포의 자살 및 노화를 유도함이 관찰되었지만, 강한 항산화 능력을 지닌 우르솔산은 세포를 저산소로부터 보호하여 정상수준으로 세포의 생존과 증식을 유지시키고, 세포자살 관련 단백질(cleaved caspase-3, Bcl-2, 그리고 cIAP) 및 세포 노화 관련 단백질(beta-galactosidase)을 조절하는 탁월한 약리학적 효과를 가지고 있었다. 따라서 본 연구결과를 통해, 우르솔산은 강력한 천연 항산화제이며, 저산소에 의해 유도된 유해 기작을 제어함으로서, 배아줄기세포의 전능성을 유지시킬 수 있는 기능성 물질이라는 것을 알 수 있었다. Ursolic acid (UA) a bio-active ingredient found in a variety of fruits and vegetables, and it has potent antioxidant activity. However, the role of UA in mouse embryonic stem (ES) cells is poorly understood. This study investigated the functional role of UA in regulating the development of mouse ES cells under hypoxia. Hypoxia did not exert a significant effect on the undifferentiated state of mouse ES cells. However, it induced reactive oxygen species (ROS) generation and increased the level of lactate dehydrogenase (LDH) production at 48 h of hypoxic exposure. Conversely, oxidative stress induced by hypoxia was significantly inhibited by UA (30 μM) pretreatment. Hypoxia significantly decreased cell survival and the level of [3H] thymidine incorporation, both of which recovered following pretreatment of UA. In addition, UA decreased the apoptotic effect of hypoxia by attenuating caspase-3 cleavage or by recovering cellular inhibition of the apoptotic protein (cIAP)-2 and Bcl-2 expression. We further found that UA decreased senescence-associated beta-galactosidase activity. We suggest that UA is a natural antioxidant and one of the functional modulators of hypoxia-induced survival, apoptosis, proliferation, and aging in mouse ES cells.

alpha 1,3-galactosyltransferase 기능 제거 및 MCP 발현 형질전환 돼지의 대동맥 혈관내피세포에 CD37/CD73 발현 세포주 개발

노진구,변승준,양현,옥선아,우제석,이휘철,황인설,김지윤,박상현,이주영,오건봉,No, Jin-Gu,Byun, Sung-June,Yang, Hyeon,Ock, Sun A,Woo, Jae-Seok,Lee, Hwi-Cheul,Hwang, In-sul,Kim, Ji-Youn,Park, Sang Hyoun,Lee, Joo Young,Oh, Keon Bong 한국수정란이식학회 2018 한국동물생명공학회지 Vol.33 No.3

Acute vascular rejection has been known as a main barrier occurring in a xenograted tissue of alpha 1,3-galactosyltransferase knock-out (GalT KO) pig into a non-human primate (NHP). Adenosine which is a final metabolite following sequential hydrolysis of nucleotide by ecto-nucleotidases such as CD39 and CD73, act as a regulator of coagulation, and inflammation. Thus xenotransplantation of CD39 and CD73 expressing pig under the GalT KO background could lead to enhanced survival of recipient NHP. We constructed a human CD39 and CD73 expression cassette designed for endothelial cell-specific expression using porcine Icam2 promoter (pIcam2-hCD39/hCD73). We performed isolation of endothelial cells (pAEC) from aorta of 4 week-old GalT KO and membrane cofactor protein expressing pig ($GalT^{-MCP/-MCP}$). We were able to verify that isolated cells were endothelial-like cells using immunofluorescence staining analysis with von Willebrand factor antibody, which is well known as an endothelial maker, and tubal formation assay. To find optimal condition for efficient transfection into pAEC, we performed transfection with GFP expression vector using four programs of nucleofection, M-003, U-023, W-023 and Y-022. We were able find that the program W-023 was optimal for pAEC with regard to viability and transfection efficiency by flow cytometry and fluorescent microscopy analyses. Finally, we were able to obtain $GalT^{-MCP/-MCP}/CD39/CD73$ pAEC expressing CD39 and CD73 at levels of 33.3% and 26.8%, respectively. We suggested that pACE isolated from $GalT^{-MCP/-MCP}$ pig might be provided as a basic resource to understand biochemical and molecular mechanisms of the rejections and as an alternative donor cells to generate $GalT^{-MCP/-MCP}/CD39/CD73$ pig expressing CD39 and CD73 at endothelial cells.

이종이식에 활용할 α1,3-galactosyltransferase 비활성화 및 Membrane Cofactor Protein 발현 동형접합 형질전환 돼지 개발

이건섭,박상현,이해선,지수정,이주영,변승준,황성수,김경운,옥선아,오건봉,Lee, Gunsup,Park, Sang Hyoun,Lee, Haesun,Ji, Soo-Jeong,Lee, Joo Yung,Byun, Sung-June,Hwang, Seongsoo,Kim, Kyung Woon,Ock, Sun A,Oh, Keon Bong 한국수정란이식학회 2017 한국동물생명공학회지 Vol.32 No.3

Transplantation is considered to be a very useful approach to improve human welfare and to prolong life-span. Heterologous organ transplantation using pig organs which are similar to human beings and easy to make mass-production has known as one of the alternatives. To ensure potential usage of the pig organ for transplantation application, it is essentially required to generate transgenic pig modifying immuno-related genes. Previously, we reported production of heterozygous ${\alpha}1,3$-galactosyltransferase (GalT) knock-out and human membrane cofactor protein (MCP) expressing pig ($GalT^{-MCP/+}$), which is enforced for suppression of hyperacute and acute immunological rejection. In this study, we reported generation of homozygous pig ($GalT^{-MCP/-MCP}$) by crossbreeding $GalT^{-MCP/+}$ pigs. Two female founders gave birth to six of $GalT^{-MCP/-MCP}$, and seven $GalT^{-MCP/+}$ pigs. We performed quantitative real-time PCR, western blot, and flow cytometry analyses to confirm GalT and MCP expression. We showed that fibroblasts of the $GalT^{-MCP/-MCP}$ pig do not express GalT and its product Gal antigen, while efficiently express MCP. We also showed no expression of GalT, otherwise expression of MCP at heart, kidney, liver and pancreas of transgenic pig. Taken together, we suggest that the $GalT^{-MCP/-MCP}$ pig is a useful candidate to apply xenotransplantation study.

Identification of the Pig β-1,3-N-acetylglucosaminyltransferase 1 (pB3GNT1) that is Involved in Poly-N-acetyllactosamine (poly-LacNAc) Synthesis

Ji-Youn Kim(김지윤),Hwan-Jin Hwang(황환진),Hak-Jae Chung(정학재),Shinichi Hochi(신이치호치),Mi-Ryung Park(박미령),Sung June Byun(변승준),Keon Bong Oh(오건봉),Hyeon-Yang(양 현),Kyung-Woon Kim(김경운) 한국생명과학회 2018 생명과학회지 Vol.28 No.4

당 단백질에 붙어 있는 당사슬 구조는 형질전환 돼지 유즙으로 분비되는 의약용 단백질의 생물학적 활성, 안정성 그리고 안전성에 영향을 줄 수 있다. 형질전환 동물을 이용한 치료용 당 단백질 생산은 유선 세포에서 이루어지는 당사슬 부가능력에 의해 제한되며, 균일한 당사슬 형태를 가지는 당 단백질 생산은 도전 과제로 남아있다. β-1,3-N-acetylglucosaminylatransferase1 (B3GNT1) 유전자는 N-아세틸글루코사민에 갈락토오스 잔기를 부착시키는 단백질 당화기작에 중요한 효소이지만, 돼지 당 전이효소에 대한 정보는 매우 제한적이다. 따라서, 돼지 B3GNT1 (pB3GNT1) 유전자를 클로닝하고 N-아세틸글루코사민에 갈락토오스 잔기를 부착시키는 기능적 특성을 조사하였다. 몇가지 다른 프라이머를 사용하여 전체 전사영역(ORF)을 함유하는 부분적인 pB3GNT1 mRNA 염기서열을 간 조직으로부터 분리하였다. 클로닝 된 pB3GNT1의 ORF는 1,248개의 뉴클레오티드를 가지며, 415개 아미노산 잔기로 구성되어 있었다. pB3GNT1 유전자의 장기별 발현특성은 성돈 및 자돈의 여러 기관에서 분석하였다. pB3GNT1 mRNA 발현 수준은 심장, 소장 보다는 근육에서 높았지만 폐에서는 낮았다. pB3GNT1의 기능적 특성 분석을 위해 돼지 신장 세포주(PK-15)에서 pB3GNT1 유전자의 안정적인 발현을 확립하였다. 그 결과, PK-15 세포에서 pB3GNT1 발현에 의한 당화 패턴은 총 시알산 증가에는 영향을 미치지 않지만, poly-N-아세틸글루코사민은 증가하는 것으로 나타났다. 본 연구는 생물반응기로 형질전환 돼지를 이용할 때 희망하는 당사슬을 부가하여 치료 가능성을 높이며 개선된 활성을 나타내는 당단백질 생산에 도움이 될 것이다. The structure of glycan residues attached to glycoproteins can influence the biological activity, stability, and safety of pharmaceutical proteins delivered from transgenic pig milk. The production of therapeutic glycoprotein in transgenic livestock animals is limited, as the glycosylation of mammary gland cells and the production of glycoproteins with the desired homogeneous glycoform remain a challenge. The β-1,3-N-acetylglucosaminylatransferase1 (B3GNT1) gene is an important enzyme that attaches N-acetylglucosamine (GlcNAc) to galactose (Gal) residues for protein glycosylation; however, there is limited information about pig glycosyltransferases. Therefore, we cloned the pig B3GNT1 (pB3GNT1) and investigated its functional properties that could attach N-acetylglucosamine to galactose residue. Using several different primers, a partial pB3GNT1 mRNA sequence containing the full open reading frame (ORF) was isolated from liver tissue. The ORF of pB3GNT1 contained 1,248 nucleotides and encoded 415 amino acid residues. Organ-dependent expression of the pB3GNT1 gene was confirmed in various organs from adult and juvenile pigs. The pB3GNT1 mRNA expression level was high in the muscles of the heart and small intestine but was lower in the lungs. For functional characterization of pB3GNT1, we established a stable expression of the pB3GNT1 gene in the porcine kidney cell line (PK-15). As a result, it was suggested that the glycosylation pattern of pB3GNT1 expression in PK-15 cells did not affect the total sialic acid level but increased the poly N-acetyllactosamine level. The results of this study can be used to produce glycoproteins with improved properties and therapeutic potential for the generation of desired glycosylation using transgenic pigs as bioreactors.

Differential Expression Patterns of Gangliosides in the Liver and Heart of NIH-miniature Pigs

Jae-Sung Ryu(유재성),Kyu-Tae Chang(장규태),Ji-Su Kim(김지수),Dong Hoon Kwak(곽동훈),Young-Choon Lee(이영춘),Keon Bong Oh(오건봉),Young-Kug Choo(추영국) 한국생명과학회 2010 생명과학회지 Vol.20 No.4

갱글리오시드는 포유동물 세포막의 중요한 구성요소로서 세포와 세포 혹은 세포와 단백질간의 상호작용을 포함한 다양한 면역학적 역할을 수행하고 있다. 이 연구는 NIH-미니돼지의 간과 심장을 인간에게 이식할려고 할 때 예측되어지는 거부 반응과 관련된 구성성분들 중 시알산음 함유하고 있는 스핑고당지질인 갱글리오시드에 대해 조사하였다. 얇은 막크로마토그래피와 면역조직화학적분석을 실시한 결과 NIH-미니돼지의 간은 갱글리오시드의 발현이 심장보다 높게 나타났다. 갱글리오시드 GD3, GDla, GDlb, GTlb는 간과 심장의 두 기관에서 발견되었다. 그러나 GQlb는 간에서만 발견되었고 심장에서는 검출되지 않았다. 이러한 결과는 갱글리오시드의 발현양상은 간과 심장에서 조직특이적이라는 것을 의미한다. 한편, GM3를 포함한 다른 갱글리오 시리즈인 갱글리오시드들은 NIH-미니돼지의 간과 심장에서 검출되어지지 않았다. 이와 같은 연구결과로부터 갱글리오시드는 미니돼지의 장기중 특히, 간과 심장의 이종장기이식과 관련된 면역거부반응에서 어떤 역할을 수행하고 있다고 여겨진다. Gangliosides are a major component of the plasma membrane of mammalian cells, which are directly involved in a variety of immunological events, including cell-to cell or cell-to-protein interactions. In this study, we investigated whether gangliosides, sialic acid-containing glycosphingolipids, are related to rejection during the xenotransplantation of NIH-miniature pig livers and hearts to humans. Both high performance thin-layer chromatography and immunohistochemistry analyses revealed that the expression of gangliosides in the liver tissue of NIH-miniature pigs was higher than that in the heart. Gangliosides GD3, GD1a, GD1b, GT1b and GQ1b were observed in both the liver and heart, whereas GQ1b was detected only in the liver, indicating that the ganglioside expression profiles are tissue specific. Moreover, other ganglio-series gangliosides, including GM3, were not detected in the livers and hearts of NIH-miniature pigs. Taken together, these results suggest that gangliosides may play important roles in immune responses in clinical xenotransplants of pig livers and hearts.