돼지 $\beta$-Casein 유전자의 3' 말단 부위의 cis-Acting Element가 유선 상피 세포내의 발현에 미치는 영향

이휘철,김병주,변승준,이승훈,김민지,정희경,이현기,조수진,장원경,박진기,이풍연,Lee, Hwi-Cheul,Kim, Byoung-Ju,Byun, Sung-June,Lee, Seung-Hoon,Kim, Min-Ji,Chung, Hee Kyoung,Lee, Hyun-Gi,Jo, Su-Jin,Chang, Won-Kyong,Park, Jin-Ki,Lee, Poong 한국동물번식학회 2008 Reproductive & developmental biology Vol.32 No.3

Tissue-specific and temporal regulation of milk protein gene expression is advantageous when creating transgenic animal that produces foreign protein into milk. Gene expression, i.e. protein production, is regulated not only by promoter strength but also mRNA stability. Especially, poly A tail length by polyadenylation affects in vivo and in vitro mRNA stability and translation efficiency of the target gene. In the present study, nucleotide sequence of 3'-UTR was analyzed to evaluate the effects of mRNA stability on the target gene expression. Based on the poly A signal of 3' -untranslated region (UTR), nucleotide sequences of putative cytoplasmic polyadenylation elements (CPEs) and downstream elements (DSEs: U-rich, G-rich, GU-rich) were analyzed and used to construct 15 luciferase reporter vectors. Each vector was transfected to HC11 and porcine mammary gland cell (PMGC) and measured for dual luciferase expression levels after 48 hours of incubation. Luciferase expression was significantly higher in construct #6 (with CPE 2, 3 and DSE 1 of exon 9) and #11 (with CPE 2, 3 and DSE 1, 2 and 3 of exon 9) than construct #1 in the PMGC. These results suggest that expression of target genes in PMGC may be effectively expressed by using the construct #6 and #11 on production of transgenic pig. 형질 전환 동물 생산에는 조직 및 시기 특이적 발현 조절이 가능하다는 장점 때문에 유즙 내로 외부 유전자를 발현시키는 시스템이 널리 이용되고 있다. 유전자 발현 즉, 단백질 생산은 프로모터의 강도뿐만 아니라 mRNA의 안정성에 의해서도 조절된다. 특히, polyadenylation에 의한 poly A의 길이는 in vivo와 올 in vitro에서 mRNA 안정성 및 목적 유전자의 번역효율에 영향을 준다. 본 연구에서는 이러한 mRNA 안정성이 목적 유전자의 발현에 미치는 영향을 알아보기 위해 3'-UTR 염기 서열을 분석하였다. 이 3'-untranslated region(UTR) 내의 poly A signal을 기준으로 putative cytoplasmic polyadenylation element(CPE) 부위와 downstream elements(DSE: U-rich, G-rich, GU-rich)의 염기 서열을 분석하고, 각각의 element를 기준으로 15 종의 luciferase reporter vector를 제작하여, 생쥐 유선 세포주(HC11)와 돼지 유선 세포주(PMGC)에 각각 transfection시킨 후 48시간 동안 배양하고 luciferase 발현량을 분석하였다. PMGC의 경우, luciferase의 발현은 exon 9의 CPE 2,3 및 DSE 1을 포함한 #6 construct에서 유의적으로 높은 발현량을 보였으며, exon 9의 CPE 2, 3과 DSE를 모두 포함하고 있는 #11 construct에서도 유의적으로 높은 발현량을 보였다. 이러한 결과는 형질 전환 돼지 생산에 있어 #6 및 11 construct의 사용은 목적의 유전자를 효과적으로 발현시키는데 기여할 것으로 사료된다.

RAPD 다형성 분석을 통한 사상체질간 유전적 상관관계에 관한 연구

이휘철(Hwi-Cheul Lee),조동욱(Dong-Wuk Cho),조중호(Joong-Ho Cho),서영우(Young-Woo Seo),이창수(Chang-Soo Lee) 대한한의학회 2000 대한한의학회지 Vol.20 No.4

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 ³ TCID₅₀/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.

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.

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.

사람 조직 플라스미노겐 활성인자 생산용 형질전환 돼지에서의 혈액학적 성상 비교

박미령(Mi-Ryung Park),황인설(In-Sul Hwang),이승훈(Seunghoon Lee),이휘철(Hwi-Cheul Lee) 한국산학기술학회 2020 한국산학기술학회논문지 Vol.21 No.12

해부학적, 생리학적으로 인간과 유사한 특성을 지닌 돼지를 이용한 실험은 의학적 분야에서 폭넓게 이용되고 있다. 돼지에서 혈액의 일반적인 성상과 이화학적 수치는 의학적 연구 및 수의학적 치료에서도 중요한 부분으로 인정되고 있으나, 형질전환 돼지에 대한 연구는 미비한 실정이다. 따라서, 본 연구에서는 htPA 형질전환 돼지의 혈액을 이용한 일반성상 및 이화학적 성상을 비교 분석하여 형질전환 돼지에 대한 기초 자료로 활용하고자 조사하였다. 일반돼지 7(LY)두와 형질전환 돼지 8(LY)두의 혈액을 각각 분석하였다. 혈액의 일반 성상은 16종을 분석하였으며, 혈청을 이용한 이화학 분석의 경우 15종 항목을 조사하였다. 그 결과 혈액의 일반 성상 분석에서는 적혈구(RBC), 평균적혈구 혈색소량(MCH)과 임파구(LYM)에서 두 그룹간 유의적 차이를 나타내었다. 이화학적 성상 분석에서는 혈중뇨소질소(BUN), 총단백질(TP), 콜레스테롤(CHOL), (ALT), 크레아틴(CREA), 감마글루타밀전이효소(GGT), 글로빈(GOB) 그리고 아밀라아제(AMYL)가 두 그룹간 유의적 차이를 나타내었다. 앞으로 지속적인 형질전환 돼지에 대한 생체정보를 조사함으로써, 기초자료로 이용할 수 있을 뿐만 아니라, 더 나아가 의학적 연구 분야에 적용 시 참고할 수 있을 것으로 여겨진다. Pigs have been used widely in biomedical research owing to their physiologic and anatomic similarities to humans. Analysis of the hematologic and biochemical values in pigs is an important basis for biomedical research and veterinary clinical diagnosis, but research on transgenic pigs has been sparse. This study was conducted to obtain basic data on transgenic pigs and to describe and compare the reference values for hematologic and biochemical parameters in human tissue plasminogen activator (htPA) transgenic pigs vs normal pigs. Blood samples were obtained from 7 normal LY (Landrace-Yorkshire crossbred) pigs and 8 transgenic pigs and 16 hematologic and 15 serum biochemical parameters were tested. Among the hematologic parameters tested, significant differences were observed in the red blood cells (RBC), mean red blood cell hemoglobin (MCH), and lymphocytes (LYM), between the non-transgenic and transgenic pigs. Among the biochemical parameters tested, the blood urea nitrogen (BUN), total protein (TP), cholesterol (CHOL), alanine aminotransferase (ALT), creatinine (CREA), gamma glutamyl transpeptidase (GGT), globin (GOB), and amylase (AMYL) showed significant differences between the two groups. Thus, the values determined in this study can be used as basic reference values for transgenic pigs and will contribute to their use in biomedical research.

RAPD 다형성 및 ABO 유전자형 분석을 통한 사상체질간 유전적 거리에 관한 연구

이휘철,조동욱,조중호,이창수 건국대학교 자연과학연구소 1999 建國自然科學硏究誌 Vol.10 No.2

본 연구는 사상체질인 태음인, 소음인 그리고 소양인간의 유전적 상관관계를 Random Amplified Polymorphic DNA(RAPD) 분석법 및 ABO 유전자형의 대립유전자의 빈도 분석을 통하여 실시하였다. 체질간 DNA 다형성을 검출할 수 있는 RAPD 분석용의 primer의 빠른 선별을 위해 체질별 혼합된 DNA 시료를 분석에 사용하였다. 일차적으로 200종류의 RAPD primer로부터 다형성을 보이는 7종류의 primer를 선별하였다. RAPD밴드로터 체질간 bandsharing(BS) 값은 0.68에서 0.71범위에 있었고, 유전적 거리는 BS 값에 의해 구했다. RAPD의 BS값에 의한 태음인과 소음인간 유전적 거리는 0.002이고, 이 두 체질과 소양인간에는 0.005로 분석되었다. 또한 체질별 ABO 유전형을 조사하여 그들의 대립유전자 빈도를 추정해 체질간 유전적 거리를 산출했다. 그 결과 태음인과 소음인사이에 유전적 거리는 0.014이고, 소양인과 나머지 두 체질간에는 0.032였다. 위와 같이 RAPD 분석법과 ABO 대립유전자의 빈도분석에 의한 체질간 상관관계는 두 방법 모두에서 태음인과 소음인이 소양인 체질보다 유전적으로 더 가깝게 나타났다. This study was carried out to be establish genetic understanding of Sasang constitutions of Teaumin, Soumin and Soyangin by Random Amplified Polymorphic DNA(RAPD) and ABO alleles frequencies analysis. We have applied RAPD analysis to pooled DNA sample as a means to achieve rapid screening of large numbers of primers for their capacity to reveal constitutions-specific polymorphisms. From initial 200 primers, 7 polymorphic primers between different constitutions were selected. The RAPD bandsharings(BS) values ranged from 0.68 to 0.71 for between three constitutions. The genetic distance between three constitutions was measured by BS values. Genetic distance by RADP analysis was 0.002 between Teaumin and Soumin, and 0.005 between Soyangin and the others. Three Sasang constitutions were investigated by the genotyping of ABO genotypes, and genetic distance was calculated from ABO allele frequencies in each constitution. As a result, genetic distance was 0.014 between Teaumin and Soumin, and 0.032 between Soyangin and the others. In conclusion, the genetic distance of Teaumin and Soumin was closer than that of Soyangin in the analysis of RAPD and ABO alleles frequencies.