Studies on the EfficientSeparation of Primordial Germ Cells and Introduction of Foreign DNA in the Chicken

정동기(Dong Kee Jeong),한재용(Jae Yong Han) 한국가금학회 1999 한국가금학회 정기총회 및 학술발표회 Vol.16 No.-

닭의 생식반월, 혈액, 그리고 원시생식기로부터 원시생식세포의 효율적 분리<br/> 본 연구는 다양한 발달단계로부터 원시생식세포를 분리하였다. 원시생식 세포가 가장 많이 분포하고 있는 일령을 선택하였다. 1일령 배자의 생식반월, 2.5일령 배자의 혈액 그리고 5.5일령 배자의 원시생식기를 이용하였다.<br/> 원시생식세포 분리 개수는 생식반월, 혈액, 그리고 원시생식기 각각 87±1.8, 103±4.0, 932±10.9개였다. 각 발달단계별 원시생식세포의 생존율은 약 70%로 비슷하였다. Ficoll방법의 개선으로 원시생식세포 분리 방법은 개선되어, 분리율은 생식반월, 혈액, 그리고 원시생식기가 각각 45±9.10%, 85±1.18%, 그리고 86±0.19%였다. 또한, 원시생식세포는 분리율을 높이기 위하여 마우스파이펫을 이용하여 개체로 분리하였다. 각기 다른 배발달 단계에 따라 분리한 원시생시세포 분리 방법은 종계의 보존 및 생식선 카이메라 생산에 유용할 것이다.<br/> 원시생식기 유래 원시생식세포 주입에 의한 외래 생식세포 확인 및 유전자 전이 형질전환 닭 생산은 유전자의 발현 및 기능을 연구하는데 매우 유용한 도구이다. 그러나 이러한 목적을 위하여 개발된 방법은 조류에 적용하기가 어렵다. 조류는 포유류와 달리 형태학적 발생학적 차이로 인하여 미세주입과 같은 방법으로 형질전환 닭을 생산하기가 매우 어렵다. 원시생식기 유래 원시생식세포의 발현과 성세포 발달은 형질전환 닭 생산에 가능성을 보여준다. 그러나 대부분의 유전자 전이가 초기 배자에서 일시적으로 발현되기 때문에 어렵다. 그리고 또한, 본 연구에서는 이렇게 유전자 전이 또는 외부에서 처리한 원시생식세포의 이동능력에 대하여 환인을 할 필요가 있었다. 그러므로, 외래 원시생식세포에 형광염색하여 주입하거나 수컷배자에 암컷 원시생식세포를 주입하여 DNA 검색하는 방법을 이용하여 외래 원시생식세포가 유전자 전이체로 사용할 수 있음을 확인하였다. 그리고 또한 전기 충격법을 사용하여 유전자를 전이 시킨 후 이를 주입하여 배자 발달단계에 따라 유전자 발현 유무를 확인하였다. 결론적으로 각기 다른 배발달 단계에서 분리된 원시생식세포 멸종 조류의 보존 및 생식선 카이메라 생산에 유용한 도구임을 확인하였고 궁극적으로 성숙된 닭의 정액에서 외래 유전자가 전이된 닭을 생산 할 수 있었다. Efficient separation of primordial germ cells from chick germinal crescent, blood and gonad<br/> This study was conducted to determine the embryonic stages for the isolation of the highest number of PGCs and to improve PGCs enrichment method. The primordial germ cells (PGCs) from different sources of chick embryos were isolated. The embryonic stage having the highest number of PGCs from each sources was selected; 1-day-old embryos for germinal crescent (stage 6-8), 2.5-day-old embryos for blood (stage 17-18) and 5.5-day-old embryos for gonad (stage 27-28). The number of PGCs from one embryonic germinal crescent, blood and gonad was about 87±1.8, 103±4.0, and 932±10.9, respectively. The viability of PGCs after Ficoll from each sources was similar, showing approximately 70%. The PGCs enrichment method was improved using Ficoll density gradient centrifugation. After this step, the purity of PGCs from germinal crescent, blood, and gonad was 45±9.10%, 85±1.18%, and 86±0.19%, respectively. Also, PGCs were picked up by mouth pipette to improve the purity. This improved method for the separation of PGCs from different sources will serve as a useful tool to preserve the foundation stocks of poultry and to produce germline chimeras.<br/> Identification and introduction of foreign DNA in the recipient chicks<br/> Expression of a foreign gene in embryos and embryonic germ cell as a gonadal PGCs would show the possibility to produce the transgenic chicken. However, transgenic chicken cannot be obtained using the transient transfection method by expression of early embryonic stage. And also, this studies must confirm the migration activity of foreign DNA transfected PGCs. Therefore, two methods of migration activity analysis were compared for the detection of exogenous PGCs. The labelled PGCs injected into 2.5-day embryos and then incubated until they reached stage 28, fluorescence was observed in the embryonic gonad of the embryos and W-specific DNA were not detected in blood and fibroblast cell of male embryos but embryonic gonad. Through this fact, gPGCs can be used as a genetic vector. In this experiment, foreign DNA was transfected into gPGCs using electroporation by Hong et al (1998). And these cells were injected into recipient embryos and estimated the efficiency of gene expression to the developmental stage, 6-day-old embryo, 10-day-old embryo and hatched chick. gPGCs transfected foreign gene producted lacZ gene were transferred sperm after sex maturation. This result indicated that gPGCs had characteristics of circulating PGCs and were useful genetic vector for the production of transgenic chicken. In conclusion, these results indicate that the improved method for the separation of PGCs from different sources will serve as a useful tool to preserve the foundation stocks of poultry and to produce germline chimeras.

Detection of Foreign Gene after Injection of Gonadal Primordial Germ Cells

정동기(Dong Kee Jeong),홍영호(Yeong Ho Hong),박태섭(Tae Sub Park),김양하(Yangha Kim Moon),한재용(Jae Yong Han) 한국가금학회 1997 한국가금학회 정기총회 및 학술발표회 Vol.14 No.-

Identification of Foreign DNA in the Chicken Genome by Gonadal Primordial Germ Cells Injection

정동기(Dong Kee Jeong),송기덕(Ki Duk Song),박태섭(Tae Sub Park),김덕경(Duk Kyung Kim),금창우(Chang Woo Kum),송원화(Gwon Hwa Song),서길주(Gil Ju Seo),한재용(Jae Yong Han) 한국가금학회 1998 한국가금학회 정기총회 및 학술발표회 Vol.15 No.-

유전 및 육종 / 생식반월 및 혈액 원시생식세포를 이용한 생식선 카이메라 닭 생산

정동기(Dong Kee Jeong),박태섭(Tae Sub Park),김덕경(Duk Kyung Kim),송기덕(Ki Duk Song),홍영호(Yeong Ho Hong),한재용(Jae Yong Han) 한국축산학회 1999 한국축산학회지 Vol.41 No.6

This study was conducted to produce germline chimeric chicken using primordial germ cells (PGCs) collected from germinal crescent (GC) and blood vessel. Korean Ogol Chicken (KOC) PGCs were collected from GC (H&H stage 6-7) and blood vessel which contains circulating PGCs (H&H stage 1617), and were injected into 2.5-day-old recipient White Leghorn (WL) embryonic blood vessel (H&H stage 16-17). The hatchabilities of manipulated embryos which were injected GC and blood PGCs were 10.7% and 23.2% respectively. Fifty one among 192 chicks arrived at sexual maturity and were used for progeny test. The progeny test for WL(KOC) by mating KOC revealed that three black feathered chicks were hatched in the group injected with germinal crescent PGCs and one black feathered chick was produced in the group transferred with blood PGCs. The black feathered chicks must have originated from the injected KOC PGCs. This result indicated that PGCs of donor KOC embryos might migrate into embryonic gonads of recipient WL and differentiate into germ cells.

지방세포 분화중인 3T3-L1 세포에서 아로마테이즈 siRNA 처리에 의한 지방관련 유전자와 전사인자의 발현 조절

정동기(Dong Kee Jeong) 한국생명과학회 2008 생명과학회지 Vol.18 No.11

본 연구는 에스트로겐 생성효소 유전자인 아로마테이즈 유전자의 siRNA를 이용하여 지방전구세포인 3T3-L1 세포의 지방세포 분화 시 나타나는 유전자의 발현을 검증하기 위하여 수행하였다. 먼저, CYP19A1 (aromatase)의 유전자로부터 siRNA를 3쌍을 디자인하고 이를 지방세포의 전구세포인 3T3-L1세포에 유전자 전이 한 후 분화 유도를 통하여 지방세포 생성의 메커니즘을 분석하였다. 결과적으로 비만의 원인 유전자인 렙틴 유전자의 발현 억제를 유도할 수 있었으며 특이적으로 인슐린과의 연관성이 매우 높음을 밝혀 낼 수 있었다. 그리고 비만 또는 백색지방 생성 시 발현이 억제되는 adiponectin과 adipsin의 과발현을 관찰할 수 있었다. 이 결과를 통하여 지방생성의 모든 신호전달체계 중 특정 한 물질을 저해 하므로써 큰 부작용 없이 비만의 문제가 되는 지방생성을 일정 정도 제어 할 수 있음을 확인 할 수 있었다. 그러므로 이 결과는 앞으로 에스트로겐 결핍 또는 과발현에 의하여 문제가 되는 지방생성 메커니즘을 밝히는 연구에 중요한 단서가 될 것으로 기대된다. This study was performed to verify the gene expression of 3T3-L1 using the siRNA of the aromatase gene, which is the estrogen synthesis enzymes. First of all three pairs of siRNA were designed from the CYP19A1 (aromatase) and analyzed the formation of fat cell mechanism by transferring gene to 3T3-L1 and differentiating it. As a result, the expression of leptin gene, which is the main gene causing the obesity, was controlled and the cause of the obesity is related with the insulin specifically. The overexpression of adiponectin and adipsin was observed. This result showed that the formation of the fat was controlled a little without any side effect by obstructing a specific material out of all the signal systems in the fat formation. This study will be an important clue to make it clear that the lack or overexpression of estrogen might be the cause of fat formation mechanism.

너도밤나무 크레오소트가 흰 쥐의 장내 미생물 변화에 미치는 영향

김정아(Jeong A Kim),유다윤(Da Yoon Yu),김인성(Jeong A Kim),이철영(Chul Young Lee),정동기(Dong Kee Jeong),이상석(Dong Kee Jeong),최인순(In Soon Choi),조광근(Kwang Keun Cho) 한국생명과학회 2018 생명과학회지 Vol.28 No.7

설사는 경제동물의 이유시기 폐사율을 일으키는 가장 높은 요인이다. 크레오소트는 전통 의약품으로 오랜 세월동안 지사제로 사용되어 왔다. 본 연구는 흰 쥐 모델에서 크레오소트 급여가 동물의 성장 효율 및 장내 미생물에 미치는 영향을 구명할 목적으로 수행되었다. 4주령의 수컷 흰쥐 24마리를 임의로 대조구, 항생제 그룹, 크레오소트 0.4% 그룹, 크레오소트 0.8% 그룹으로 배치하였다. 대조구는 기초사료, 항생제 그룹은 apramycin 0.5%, 크레오소트 그룹은 크레오소트 0.4%와 0.8% 수준으로 하여 예비시험 기간 1주일, 본시험 기간 4주일 동안 급여하였다. 일당증체량은 실험구간 차이가 없었으나, 사료 섭취량은 Creo 0.8 그룹에서 유의적으로 증가하였다(p<0.05). 장내 미생물에 대한 문(phylum) 수준 분석 결과 Creo 0.8 그룹에서 Firmicutes가 감소하고 Bacteroidetes가 증가하여 F/B 비율을 감소시키는 것으로 나타났다(p<0.05). 과(family) 수준에서는 Lachnospiraceae가 크레오소트 0.8% 수준에서 증가되었으며(p<0.01), 속(genus) 수준에서는 Turicibacter가 감소되었다(p<0.01). 종(species) 수준에서는 Clostridium disporicum이 감소되었다(p<0.01). 이상의 결과는 쥐에 크레오소트 급여는 사료 섭취량을 증가시키고 장내 미생물의 변화에 영향을 미칠 수 있다는 것을 시사한다. Diarrhea is one of the main disorders which cause the highest level mortality of the post-weaning economic animal. Beech-wood creosote has been used as a traditional anti-diarrheic medicament for a long time. The present study was conducted to investigate the effects of dietary supplementation of Beech-wood on growth performance and intestinal microbiota in rats. Twelve 4-week-old rats were randomly assigned to one of four dietary groups and fed a basal diet supplemented with none (CON), 0.5% apramycin (ANTI), 0.4% creosote (Creo 0.4), or 0.8% creosote (Creo 0.8) for 4 weeks following 1 week of adaptation period to the respective diet. Average daily gain was not influenced by the dietary treatment whereas average daily feed intake was greatest for the Creo 0.8 group. In the intestinal microbiota at the level of the phylum, the percentage of Firmicutes bacteria decreased but Bacteroidetes increased in the Creo 0.8 group vs. Control, which resulted in a decreased F/B ratio for the former (p<0.05). Moreover, the percentage of Lachnospiraceae was greater at the level of the family for the Creo 0.8 group than for Control, but the percentages of Turicibacter and Clostridium disporicum were less in the former (p<0.01) at the genus and species levels, respectively. Collectively, the present results indicate that dietary supplementation of creosote increases the feed intake and also influence the intestinal microbiota in rats.

제주재래흑돼지×Landrace F₂ 집단의 성장형질에 대한 교배조합의 효과

강용준,조상래,정동기,이재봉,박희복,조인철,한상현,Kang, Yong-Jun,Cho, Sang-Rae,Jeong, Dong Kee,Lee, Jae-Bong,Park, Hee-Bok,Cho, In-Cheol,Han, Sang-Hyun 한국수정란이식학회 2016 한국동물생명공학회지 Vol.31 No.1

This study was conducted to examine effects of mating type on the growth traits in an $F_2$ population produced by reciprocal intercrosses between Landrace and the Jeju Native Black pig (JBP). The $F_2$ progeny were produced by two different mating types based on the grand dams of founder breeds JBP (Cross_1) and Landrace (Cross_2). The body weights at 21 days after birth (BW21D) was significantly different between Cross_1 and Cross_2 (P<0.05), showing that the BW21D of Cross_1 has about 0.25 kg heavier than Cross_2. The significant differences were found between males and females for the growth traits including the body weights (BWB, BW21D, BW70D and BW140D) and average daily gains (ADG, eADG and lADG) (P<0.05). Males were heavier BWB, BW21D and BW140D levels, and higher ADG and lADG levels than females. On the other hand, females had heavier BW70D and higher eADG levels than those of males. When considering the mating types and sex simultaneously the Cross_2 males had the heaviest BW140D among the combinations of cross and sex. In conclusion, it is desirable to choose Landrace as grand dams in the reciprocal intercrosses between Landrace and JBP for producing their progeny construction and to plan the production of $F_2$ males for industrial purposes. These results suggested that it may be one of useful strategies to improve the productivity through out selection of the mating type of founder breeds and the progeny sex, especially in Landrace, JBP and their related populations.

OCX-32 유전자 내 c.494A>C 및 c.267T>G SNP이 한국 재래닭 산란형질에 미치는 효과 분석

이지연,최소영,김종대,홍영호,정동기,이성진,Lee, Ji-Yeon,Choi, So-Young,Kim, Chong-Dae,Hong, Yeong Ho,Jeong, Dong Kee,Lee, Sung-Jin 한국가금학회 2014 한국가금학회지 Vol.41 No.3

The identification and utilization of potential candidate genes with significant effects on economically important traits have become increasingly important in poultry breeding programs. The ovocalyxin-32 (OCX-32) gene is located chromosome 9 in chicken, plays an important role in eggshell formation. This study was performed to assess the association between single nucleotide polymorphisms (SNPs) of OCX-32 gene and egg production traits in the Korean native chicken. Four Korean native chicken population (n = 181; including 46 females of Ogol, 46 females of white, 43 females of gray and 46 females of black) were used to analyze two SNPs (c.494A>C and c.267T>G) in the OCX-32 gene by PCR-RFLP (Polymerase Chain Reaction-Restricted Fragment Length Polymorphism). We measured egg production traits of age at first egg, first egg weight, egg production ratio and egg weight. The SNP c.494A>C was significantly associated with egg production ratio in Korean Ogol chickens (p<0.001) and egg weight in Korean white chickens (p<0.05). SNP c.267T>C was significantly associated with egg weight in Korean Ogol chickens (p<0.05). But there was no significant association in Korean gray and black chickens. Results suggest the possibility of using molecular markers in OCX-32 gene as a tool for performance and egg production traits in Korean native chicken breeding program.

Production of Germline Chimeric Chickens by Transfer of Cultured Primordial Germ Cells

홍영호(Yeong Ho Hong),정동기(Dong Kee Jeong),박태섭(Tae Sub Park),김양하(Yangha Kim Moon),한재용(Jae Yong Han) 한국가금학회 1997 한국가금학회 정기총회 및 학술발표회 Vol.14 No.-

Association between Genotypes of the Isocitrate Dehydrogenase 3, beta subunit (IDH3B) Gene and Carcass Traits in an F₂ Crossbred Population of Landrace × Jeju (Korea) Black Pigs

Yong-Jun Kang(강용준),Dong Kee Jeong(정동기),In-Cheol Cho(조인철),Sang-Hyun Han(한상현) 한국생명과학회 2016 생명과학회지 Vol.26 No.4

본 연구에서는 제주흑돼지와 랜드레이스 품종 사이에서 생산된 F₂ 교배 집단의 경제형질과 isocitrate dehydrogenase 3, beta subunit (IDH3B) 유전자의 유전적 다형성의 상관관계를 시험하였다. IDH3B 유전자의 프로모터 지역에서 304-bp 삽입/결실 돌연변이를 기준으로 전체 1,105 F₂들에 유전자형 분석에 이용하였다. 기초축군과 F₁, F₂에서 세 가지 유전자형(AA, AB, BB)이 모두 발견되었다. 통계적 상관 분석결과에서 도체중(CW), 세 지점(4-5번 흉추사이, 11-12번 흉추 사이, 13번 흉추-1번 요추 사이)에서 측정한 등지방두께와 도체장(CL)의 수준은 유전자형애 따른 유의적인 차이를 나타내었지만(p<0.05), 육색(MC), 등심단면적(EMA)과 근내지방도(MARB)은 유의적인 차이를 나타내지 않았다(p>0.05). IDH3B 동형접합자 BB를 보유한 F₂ 돼지들은 도체중이 더 무겁고(80.790±0.725 kg), 도체장은 더 짧은(101.875±0.336 cm) 양상을 보였다(p<0.05). 또한, IDH3B 유전자형 BB인 개체들은 IDH3B AA나 AB 유전자형에 비해 4-5번 흉추 사이, 11-12번 흉추 사이에서 측정된 등지방두께도 더 두꺼운 수준을 나타내었다 (p<0.05). 이상의 결과들은 IDH3B의 유전적 다양성이 제주흑돼지와 랜드레이스와 관련된 교배육종 체계의 산육능력 향상을 위한 유전적 분자 표지인자로 활용될 수 있음을 나타내었다. This study tested the association between genetic polymorphisms of the isocitrate dehydrogenase 3, beta subunit (IDH3B) gene and economic traits in an F₂ crossbred population of Landrace × Jeju (South Korea) Black pigs. A 304-bp insertion/deletion mutation in promoter region was screened for determining genotypes of the IDH3B gene in a total of 1,105 F₂ pigs. Three genotypes (AA, AB, and BB) were identified in the founder, F₁, and F₂ populations. Association analysis showed significant differences in carcass weights (CW), backfat thicknesses in three positions of the body (4<SUP>th</SUP>-5<SUP>th</SUP> ribs, BF5; 11<SUP>th</SUP>-12<SUP>th</SUP> ribs, BF12; 13<SUP>th</SUP> rib-1<SUP>st</SUP> lumbar, BFL), and carcass lengths (CL) (p<0.05), but not in meat color (MC), eye muscle area (EMA), or marbling scores (MARB) (p>0.05). The F₂ IDH3B BB homozygotes showed heavier CW (80.790±0.725 kg) and shorter CL (101.875±0.336 cm) than the other genotypes (p<0.05). In addition, the BF levels between the 4<SUP>th</SUP> - 5<SUP>th</SUP> and 11<SUP>th</SUP> - 12<SUP>th</SUP> vertebrae were thicker in the carcasses of pigs with the IDH3B BB genotype than with the other genotypes (p<0.05). These results suggested that genetic variations in the IDH3B gene may serve as molecular genetic markers for improving the Landrace × Jeju Black pig crossbreeding systems.