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Production of Recombinant Human Von Willebrand Factor in the Milk of Transgenic Pigs
LEE, Hyun-Gi,LEE, Hwi-Cheul,KIM, Sung Woo,LEE, Poongyeon,CHUNG, Hak-Jae,LEE, Yun-Keun,HAN, Joo-Hee,HWANG, In-Sul,YOO, Jong-Il,KIM, Yong-Kook,KIM, Hun-Taek,LEE, Hoon-Taek,CHANG, Won-Kyong,PARK, Jin-Ki Society for Reproduction and Development 2009 The Journal of reproduction and development Vol.55 No.5
<P>Von Willebrand factor (vWF), a large multimeric glycoprotein present in blood plasma, is a blood protein of the coagulation system. It is defective in von Willebrand disease and is involved in a large number of other diseases, including thrombotic thrombocytopenic purpura-hemolytic uremic syndrome and heyde's syndrome. We have developed a line of transgenic swine harboring recombinant human von Willebrand factor (rhvWF) cDNA through microinjection of fertilized one-cell pig zygotes. Expression of rhvWF in the mammary gland and secretion of rhvWF into the milk of the transgenic swine were confirmed by immunohistochemical and western blot analyses, respectively, and rhvWF proteins were detected in milk from all lactating founder females at concentrations that were 28- to 56-folds greater than that in circulating human plasma. The amino acid sequence of rhvWF protein in the transgenic pig milk matched that of vWF produced from human blood plasma. This study provides evidence that production of rhvWF from transgenic pig milk is a potentially valuable technology and can be used as a cost-effective alternative in clinical applications.</P>
Lee, Hyun-Gi,Seong, Hwan-Hoo,Im, Seok-Ki,Chung, Hee-Kyoung,Lee, Poongyeon,Lee, Yeun-Kun,Min, Kwan-Sik,Chang, Won-Kyoung,Lee, Hoon-Taek 한국수정란이식학회 2002 한국수정란이식학회 학술대회 Vol.2002 No.1
Human eryhropoietin (EPO) is acidic glycoprotein hormone that plays key role in hematopoiesis by facilitating differentiation of erythrocyte and formation of hemoglobin (Hb) and is used for the treatment of anemia. Human EPO is consist of 166 amino acids which is modified by three N-glycosylations (24, 38, 83) and single O-glycosylation (126). N-glycosylation is reported to be related to the cellular secretion and activity of EPO. In this study, we examined effects of mutagenesis in glycosylation site of recombinat hEPO for the cellular secretion during production from cultured CHO cell. We produced rhEpo which was cloned by PCR from human liver cDNA (TaKaRa) in cultured CHO cell. Using supernatant of the culture, ELISA assay and western analysis were performed. To estimate biological activity, 20IU of rhuEpo was subcutaneously injected into four ICR mice. After 8 days, HCT level was increased average 13 per cent, RBC was increased ca. 2106//. In disease model Rat (anemia c-kit, WSRC-WS/WS), HCT was increased ca. 12%, RBC was increased ca. 1.6106//. These results suggests that rhEpo we produced has biological activity. To remove glycosylation site by substituting 24, 38, 83, and 126th asparagine (or serine) with glutamic acid, overlapping -extension site-directed mutagenesis was performed. To add novel glycosylation sites, 69, 105th leucine was mutated to asparagine. Mutant EPO construct was transfected into CHO cell. Supernatant of the cell culture was analyzed using ELISA assay with monoclonal anti-EPO antibody (Medac, Germany). Since, several reports for mutagenesis of glycosylation sites showed case-by-case results, we examined both transient expression and stable expression. Addition of novel glycosylation sites resulted no secretion while deletion mutants had little effect except some double deletion mutants (24/83 and 38/83) and triple mutant. We suggest that not single but combination of glycosyl group affect secretion of EPO.
Cloning and Characterization of Bovine 5-Cytosine DNA Methyltransferase I cDNA
Poongyeon Lee,Kwan-Sik Min,Hyun-Gi Lee,Soon-Jeung Kim,Hee-Kyoung Chung,Myung-Kyu Seo,Yun-Keun Lee,Sung-Woo Kim,Jin-Ki Park,Hwan-Hoo Seong,Moosik Kwon,Won-Kyong Chang 한국동물생명공학회(구 한국동물번식학회) 2003 Reproductive & developmental biology Vol.27 No.1
Cloning and Characterization of Bovine 5-Cytosine DNA Methyltransferase I cDNA
Poongyeon Lee,Kwan-Sik Min,Hyun-Gi Lee,Soon-Jeung Kim,Hee-Kyoung Chung,Myung-Kyu Seo,Yun-Keun Lee,Sung-Woo Kim,Jin-Ki Park,Hwan-Hoo Seong,Moosik Kwon,Won-Kyong Chang 한국동물번식학회 2003 Reproductive & Developmental Biology(Supplement) Vol.27 No.1s
Study on the Reproductive Function in Transgenic Pig Harboring Human Erythropoietin (hEPO) Gene
Hyun-Gi Lee,Hwi-Cheul Lee,Hak-Jae Chung,In-Sul Hwang,Myoung-Seob Choi,Sung-June Byun,Seunghoon Lee,Min-Ji Kim,Jae-Seok Woo,Won-Kyong Chang,Poongyeon Lee,Hoon-Taek Lee,Jin-Ki Park 한국동물생명공학회(구 한국동물번식학회) 2008 Reproductive & developmental biology Vol.32 No.2
Our previous study showed that transgenic (TG) pigs harboring human EPO (hEPO) gene have been shown to have reproductive disorders, including low pregnancy rates, irregular estrus cycle and low little size. To investigate these reasons, we assessed estrus behavior (standing response) and plasma 17B-estradiol (E2) level, which partly reflect reproductive function, during the estrus cycles after synchronization and superovulation by hormone treatments. Then, we analysed blood composition and expression of hEPO gene in TG pigs. Pigs were injected with PG600. After 10 days, pigs were fed with Regumate porcine for 6 days. Blood samples were collected from jugular vein. Analysis of blood composition and E2 level were measured by Hemavet 950 and E2 ELISA kit, respectively. And, the expression of hEPO gene in reproductive organs was quantitated by real-time RT-PCR. The percentage of estrus behavior in TG was significantly decreased. Hematocrit (HCT), hemoglobin (Hb) concentration and red blood cell (RBC) number were significantly higher in TG than wild type (WT). On the other hand, high expression of hEPO gene in TG was observed in the mammary gland as well as in the uterus. Moreover, plasma E2 level was significantly higher in TG than WT. These results suggest that nonspecific expression of hEPO gene in the other organs of TG may affect blood composition and plasma E2 level, thereby causing reproductive disorders.
3D8 scFv 형질전환 돼지 개발 및 PRRS 저항성 평가
이휘철 ( Hwi-cheul Lee ),이건섭 ( Gunsup Lee ),김지윤 ( Ji-yoon Kim ),양현 ( Hyeon Yang ),이보람 ( Bo Ram Lee ),박미령 ( Mi-ryung Park ),황인설 ( In-sul Hwang ),이풍연 ( Poongyeon Lee ),변승준 ( Sung-june Byun ),김원일 ( Won-il K 한국동물위생학회(구 한국가축위생학회) 2020 韓國家畜衛生學會誌 Vol.43 No.4
In this study, we have developed 3D8 scFv transgenic pig (TG) by microinjection of fertilized one-cell pig zygotes (2.17%). The effect of 3D8 scFv TG on porcine reproductive and respiratory syndrome virus (PRRSV) resistance were evaluated through PRRSV VR2332 (1×10 <sup>3</sup> TCID<sub>50</sub>/mL) challenge and transmission experiments. As a result, the average daily weight gain (ADWG) of TG increased compared to the wild type pigs (WT) in PRRSV challenge groups and the serum viremia levels of the TG was significantly lower than of WT on the 7 day and 21 day after infection, meaning that the viral shedding was suppressed by 3D8 scFv expression. These results suggest that the expression of 3D8 scFv in pig could suppress spreading of infected virus to pigs sharing a room.
Gene Cloning and Nucleotide Sequence of Human Dihydrolipoamide Dehydrogenase-Binding Protein
Lee, Jeongmin,Ryou, Chongsuk,Jeon, Bong Kyun,Lee, Poongyeon,Woo, Hee-Jong,Kwon, Moosik Asian Australasian Association of Animal Productio 2002 Animal Bioscience Vol.15 No.3
The pyruvate dehydrogenase complex (PDC), a member of $\alpha$-keto acid dehydrogenase complex, catalyzes the oxidative decarboxylation of pyruvate with the formation of $CO_2$, acetyl-CoA, NADH, and $H^+$. This complex contains multiple copies of three catalytic components including pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3). Two regulatory components (E1-kinase and phospho-E1 phosphatase) and functionally less-understood protein (protein X, E3BP) are also involved in the formation of the complex. In this study, we have partially cloned the gene for E3BP in human. Nine putative clones were isolated by human genomic library screening with 1.35 kb fragment of E3BP cDNA as a probe. For investigation of cloned genes, Southern blot analysis and the construction of the restriction map were performed. One of the isolated clones, E3BP741, has a 3 kb-SacI fragment, which contains 200 bp region matched with E3BP cDNA sequences. The matched DNA sequence encodes the carboxyl-terminal portion of lipoyl-bearing domain and hinge region of human E3BP. Differences between yeast E3BP and mammalian E3BP coupled with the remarkable similarity between mammalian E2 and mammalian E3BP were confirmed from the comparison of the nucleotide sequence and the deduced amino acid sequence in the cloned E3BP. Cloning of human E3BP gene and analysis of the gene structure will facilitate the understanding of the role(s) of E3BP in mammalian PDC.
초위성체 표지로 본 한국 재래닭 집단의 분자유전학적 구성
이풍연(Poongyeon Lee),연성흠(Seong-Heum Yeon),김재환(Jae-Hwan Kim),고응규(Yeoung-Gyu Ko),손준규(Jun-Kyu Son),이희훈(Hee-Hoon Lee),조창연(ChangYeon Cho) 韓國家禽學會 2011 韓國家禽學會誌 Vol.38 No.2
초위성체(MS) 표지를이용하여한국재래닭집단의각각의 분자유전학적 특성을 조사하고, 그 평가를 통해 한국 재래닭에 대한 품종 및 계통 분류의 기초를 마련하고자 본 연구를 수행하였다. 또한, 한국 재래닭 집단 내 및 집단간 유전적 변이성을 확인하고, 그 분류 및 특성 평가를 위한 MS 분석 체계를 마련하여 국내 가축유전자원의 관리에 활용코자 하였다. 국내 관리 기관 및 농가 보유 11개 계통의 한국 재래닭 및 상용계 462 수를 대상으로 19개 MS 표지로 분석한 결과, 한국 재래닭 집단은 상용계부터 분자유전학적으로 별개의 집단으로 구분되며, 특히 한국 재래닭 중 긴꼬리닭 계통은 상용계와 국내 토종닭 어느 집단과도 확연히 분리되는 것을 확인하였다. 한국 재래닭 집단 간의 유전거리는 0.11~0.18로 비교적 낮게 나타났으나, 유전적 균일도는 R 계통을 제외하고 0.86~0.88로 코니쉬 계통을 제외한 상용계의 0.95~0.97보다 비교적 낮았다. 다만, 긴꼬리닭 집단의 유전적 균일도는 0.91~0.97로 높게 나타났다. 본 연구를 통하여 한국 재래닭 집단 간의 유전적 차이 및 동질성, 그리고 집단내 의유전적 균일성을 확인하고, 긴꼬리닭 계통의 위치를 확인하였다. 이러한 결과는 국내 유전자원의 고유성을 인정할 수 있는 과학적인 근거로서, 국가 수준의 가축유전자원 평가, 관리의 기초자료로 활용될 수 있을 것이다. The study was conducted to select and optimize microsatellite (MS) markers for evaluate Korean Native Chicken (KNC) breeds in order to provide standard for the classification and breed definition of the indigenous breeds. The study also aimed to characterize and classify each KNC populations for inventory and management of avian genetic resources. A total of 462 chickens from 11 populations of chicken breeds including eight KNC breeds and three commercial chicken breeds were analyzed with 19 MS markers. KNC breeds, especially Long-Tail Chicken breeds, formed separate cluster from those commercial chicken breeds. Genetic distances between KNC populations (0.11~0.18) were relatively shorter. Genetic uniformity of KNC (except KNCR breed) (0.86~0.88) were higher than that of commercial breeds (except Cornish) (0.95~0.97). On the other hand, genetic uniformity of KNC Long Tail (KNCLT) were relatively higher (0.91~0.97). The result can be used to evaluate and manage animal genetic resources at national scale.