동결보존된 생쥐 미세조작배의 체외발생에 관한 연구

전익수(I . S . Jeon),김선의(S . E . Kim),정진관(J . K . Jung),정일정(I . C . Cheong),박영식(Y . S . Park),최광수(K . S . Choi) 한국축산학회 1993 한국축산학회지 Vol.35 No.6

The effect of micromanipulation and zona pellucida(ZP) on in vitro viability and development of frozen embryos was investigated for the effective cryopreservation of biopised embryos. The ZP-intact 4-cell embryos and ZP-removed 4-cell embryos were biopised using micromanipulator and micropipette, respectively. Biopsied embryos were equilibrated in M16 containing 3.5M DMSO and 0.25M sucrose for 1.5 minutes and then were ultrarapidly frozen in LN₂. The frozen embryos were thawed at 37℃ and were cultured in M 16 at 37℃ for 48 hours under an atmosphere of 5% CO₂ and 100% humidity. The developmental rate of fresh and frozen embryos was evaluated as percentage of embryo development from 4 cell stage to blastocyst. The results were summarized as: 1) the survival rate of 4-cell embryos ultrarapidly frozen and thawed was 73%. The development rate of survived embryos after freezing was 92%; 2) the survival rate of micromanipulated-embryos ultrarapidly frozen and thawed was 79?. The development rate of survived embryos after freezing was 59%; 3) the developmental rate of the cultured fresh ZP-intact embryos was significantly higher than those of the fresh ZP-removed embryos and fresh ZP-removed biopsied embryos (p$lt;.05); and 4) the developmental rate of the cultured frozen ZP-intact embryos was significantly higher than those of the frozen ZP-removed embryos and frozen ZP-removed biopsied embryos(p$lt;.05).

사람의 SOD-3 단백질을 발현하는 형질전환 닭 생산 연구

변승준(S. J. Byun),박철(C. Park),김진아(J. A. Kim),우제석(J. S. Woo),이휘철(H. C. Lee),김태윤(T. Y. Kim),김상훈(S. H. Kim),성환후(H. H. Seong),박진기(J. K. Park),전익수(I. S. Jeon) 한국가금학회 2008 韓國家禽學會誌 Vol.35 No.3

리포좀을 이용한 형질전환 닭 생산에 대한 연구

변승준,박철,양보석,김태윤,손시환,김상훈,전익수,Byun S. J.,Park C.,Yang B. S.,Kim T. Y.,Sohn S. H.,Kim S. H.,Jeon I. S. 한국가금학회 2004 韓國家禽學會誌 Vol.31 No.4

본 연구는 기존의 형질전환 닭 생산방법 중의 하나인 1세 포기 수정란에 유전자를 직접 주입하는 유전자 미세주입방법을 개선할 목적으로 리포좀과 외래 표지 발현유전자인 GFP를 사용하여 외래유전자의 핵전이 효율성과 주입된 외래 유전자의 발현의 지속성을 닭의 배자에서 검증하고자 시도하였다 외래유전자는 배반엽 단계 혹은 1세포기 수정란의 세포질에 리포좀과 유전자의 혼합물 혹은 오직 유전자만을 미세주입을 하였다. 연구 결과들은 리포좀을 사용한 경우 naked DNA에 비하여 배반엽 단계와 1세포기 수정란 모두에서 효율적으로 외래 유전자를 핵내로 도입할 수 있음을 배양 3과 4일차 닭의 배자에서 GFP발현 양상을 통하여 확인하였다. 또한 주입된 외래 유전자에 의해 만들어진 GFP는 배자에서 일주일 정도 지속적으로 발현됨이 관찰되었다. 리포좀 방법은naked유전자 주입 방법에 비해 1세포기와 배반엽 단계 수정란 모두에서 효율적으로 외래 유전자를 핵내로 이동시키는 능력을 가지나, 주입된 유전자의 염색체 삽입에는 큰 영향을 미치지 않는 것으로 판단된다. 따라서 닭의 수정란에서 리포좀 방법은 외래유전자 도입에 유용한 수단으로 이용되어질 수 있을 것으로 사료된다. Microinjection of DNA is a general method for generating transgenic animals, but the rate of transgenesis in chickens is very low. So it was carried out to investigate the efficiency of liposome-mediated gene transfer in stage one cell of chicken embryo with GFP expression vector. In order to determine efficiency and duration of the introduced foreign gene, it was microinjected DNA with liposome or naked DNA into the germinal disc of stage one cell or stage-X chicken embryo. Analysis of reporter gene expression in day-4 embryos showed that GFP expression was observed only in the liposome-mediate embryo groups and detectable up to day-8 embryos. The results suggest that stable integration of the introduced gene using liposome is a rare event. Nevertheless the liposome-mediated gene transfer may be a useful method to transfer a foreign gene into the stage one cell of chicken embryos.

한국 재래닭의 주령별 각 조직의 텔로미어 함량과 텔로머레이스 활성도 분석

정길선,조은정,최덕순,이민정,박철,전익수,손시환,Jung G.S.,Cho E.J.,Choi D.S.,Lee M.J.,Park C.,Jeon I.S.,Sohn S.H. 한국가금학회 2006 韓國家禽學會誌 Vol.33 No.2

텔로미어는 염색체를 보호하고 세포 분열의 안정성에 주된 작용을 하며 세포의 사멸, 노화 및 암의 발생과 직접적 관련이 있다고 알려져 있다. 최근 텔로미어의 길이와 텔로머레이스의 활성에 대한 많은 연구들은 광범위하게 진행되어 왔지만 닭에서는 매우 제한적으로 연구되어왔다. 따라서 본 연구에서는 한국 재래닭에서 발육, 성장 및 노화 단계별 간, 뇌, 심장, 신장, 정소 및 백혈구 세포에 대한 텔로미어의 양적 분포와 텔로머레이스 활성도를 분석 고찰하고자 하였다. 텔로미어의 함량 분석은 telomeric DNA probe 를 이용하여 Q-FISH 법으로 수행하였고, 텔로머레이스 활성도 분석은 TRAP 방법을 이용하였다. 분석 결과, 닭 염색체상 텔로미어는 모든 염색체 양 말단부에 나타나며 특히 1, 2 및 3 번 염색체에서는 양 말단 외 interstitial telomeric DNA 가 존재하였다. 닭의 조직별 세포들의 telomeric cDNA 함량을 분석한 결과 성장 및 노화가 진행됨에 따라 대부분의 세포들에서 텔로미어 함유율이 유의적으로 감소하였고, 조직 간 텔로미어 함유율 에서도 많은 차이를 보였는데 특히 증식성 세포인 정소 내 세포들이 다른 비 증식성 세포들에 비해 월등히 높게 나타났다. 텔로머레이스 활성도는 간, 뇌, 심장 등 대부분의 조직에서 성장 및 노화가 진행됨에 따라 활성이 감소되거나 없어지나 생식선 조직인 정소세포는 연령과 무관하게 지속적으로 높은 활성을 나타내었다. 이상의 결과로부터 닭의 조직별 세포 분화 및 증식성 특이성과 텔로미어의 함량 및 텔로머레이스 활성도 간에는 매우 밀접한 관련이 있으며, 텔로머레이스 활성도와 텔로미어 함유율 간에 매우 높은 상관이 있었다. Telomeres are essential for chromosome stability and are related with cell senescence, apoptosis and cancer. Even though telomere length and telomerase activity have been studied extensively, very little is known to analyze the telomere dynamics in chicken cells. This study was carried out to analyze the telomere distribution and telomerase activity of Korean Native Chicken cells along with aging. The cells were collected from brain, heart, liver, kidney and germinal tissues during physiological stages. Telomere distribution was analyzed by Quantitative-Fluorescence in situ Hybridization (Q-FISH) techniques using the chicken telomeric DNA probe. Telomerase activity was performed by Telomeric Repeat Amplification Protocol (TRAP) assay. In results, the telomeres of chicken were found at the ends of all chromosomes with the interstitial telomeres on chromosomes 1, 2 and 3. The amount of telomeres on chicken cells was decreased along with aging in most tissues. Furthermore, the telomere quantity was significantly different among tissues. The relative amount of telomeres in proliferous cells such as testis cells had much more than those of liver, brain, heart, blood and kidney cells. The telomerase activity was down-regulated in cells of brain, heart and liver tissues. Whereas gonadal cells showed a constitutive activity of telomerase during all stage of life. In conclusions, the telomere quantity and telomerase activity in chicken are closely relate to cell proliferation and tissue specificity during developmental stages and aging. There is also closely correlated between the amounts of telomeric DNA and telomerase activity in chicken tissues.

1 세포기 닭 수정란의 체외배양과 대리난각 배양 기술의 개선

전익수 한국동물자원과학회 2000 한국축산학회지 Vol.42 No.6

To improve the chicken embryo development rate of in vitro culture and surrogate egg-shell culture, and percentage of embryo collection, the effect of donor hens and quality of embryos were studied. In this study, the embryo collection rates were 86.8% for commercial brown layers and 77.1% for Korea Native Chicken, respectively. The difference was significant between the two breeds(p$lt;0.05). For commercial layers, the embryo development rate by in vitro culture and surrogate egg-shell culture for different regions of magnum of donor hens tended higher at 10 ㎝ to 14 ㎝ of magnum than other regions. However, for Korea Native Chicken the highest embryo development rate was found at 5 ㎝ to 9 ㎝. For efficient collection of single cell stage embryo, high yielding commercial layers are recommended to be selected as experimental chick. The donor layers must have a regular ovulation time and have laying at the day of egg collection. To increase the embryos, we suggest the process of 1st and 2nd culture of exchanging culture media and vessel is not necessary.

메추리 수정란의 대리난각 배양

최광수,전익수,손시환,오노타마오 한국축산학회 1997 한국축산학회지 Vol.39 No.1

This study examined the pattern of in vitro development of quail embryos cultured in surrogate egg-shells and investigated an in vitro culture system of quail embryos to provide opportunities for generic manipulation of quails. To investigate die pattern of in viro development of quail embryos in surrogate egg-shells, 2-day-old quail embryos were implanted into surrogate egg-shells, sealed with wrap and cultured at 37.6℃ with relative humidity of 65.5±5%. Quail embryos in the blasfodermal stage were implanted into surrogate quail and chicken egg-shells sequentially, sealed with wrap, and cultured at 37.6℃ with the relative humidity of 65.5±5%. An average length of the third toe of quail embryos cultured in surrogate egg-shells at 15 days of incubation was 12.49㎜, and was 13.03㎜ for the control culture. An average dry weight of quail embryos cultured in surrogate egg-shells at 15 days of incubation was 1.216g, and was 1.042g for the control culture. When blastodermal embryos were cultured in surrogate egg-shells of a chicken, the average hatchabilities were 11.8% for wild type and 24.6% for black type. When two-day-old embryos were cultured in surrogate egg-shells of the chicken, average hatchabilities were 55.6% for wild type and 51.3% for black type, and when blastodermal embryos were cultured in surrogate eggshells of quail from the blastodermal stage to 2-days-old, and thereafter to hatching were recultured in surrogate egg-shells of chickens, average hatchabilities were 36.0% for wild type and 51.9% for black type. The results for our culture system indicated that it would be practical for the production of transgenic birds.

닭 - 메추리 속간잡종의 특성과 핵형 연구

최광수,전익수,손시환,정일정 한국축산학회 1997 한국축산학회지 Vol.39 No.1

The characteristics and a karyological study of chicken-quail hybrids were investigated. Fertility of the genus-crossing between chickens and quail was 18.3%, among which the hatchability was 4.9%. From the chromosomal analysis of the genuscrossbred between chickens and quail, 55.6% of the embryos were males and the ones that hatched were all males, Average body weights of the genus-crossbred between chickens and quail were 197.8g at 8 weeks of age and 329.9g at 12 weeks of age, which were heavier than those of male quails of 128.2g and 126.4g at similar ages. The growth of the genus-crossbred was much faster than that of quails after 6 weeks of age. The origin of chromosomes 1, 2, and 4 from embryos and leukocytes of the chicken-quail hybrids was distinguished from chicken and quail. Differences of morphological features were not significant among tissues.

배반엽 세포 주입에 의한 닭 - 메추리 키메라 생산

최광수,전익수,손시환,오노타마오 한국축산학회 1997 한국축산학회지 Vol.39 No.1

This study examined the production of chick-quail chimera by transfer of blastoderm cells and investigated the developmental pattern of chick blastoderm cells in the quail subgetminal cavity. To produce the chick-quail chimera, 1,500 to 2,000 cells of chick embryos in the blastodermal stage were microinjected into the subgerminal cavity of quail embryos. The presence of the blastoderm cells of chicks in the subgenninal cavity of quail embryos was detected by Feulgen staining. The developmental pattern of the blastodenn cell division of chicks in the quail subgerminal cavity was examined by chromosomal analysis. When quail embryos which were injected with chick blastoderm cells were cultured in surrogate egg-shells, average hatchabilities were 40.0% for the wild type and 45.8% for the black type. A chick-quail chimera was produced from the black type. After the blastoderm cells of chicken embryos were itgected into the subgeminal cavity of quail embryos, the presence of blastoderm cells of chicken embryos was detected by Feulgen staining in the ectoderm, endoderm, and mesoderm of the subgetminal cavity of quail embryos. When the blastoderm cells of chicken embryos were injected into the subgemunal cavity of quail embryos, the chromosomal analysis revealed that the occurrence of blastoderm cell division of chicken embryos was exxremely rare.

미세현미조작된 생쥐배의 발생과 단일할구의 성감별 기술에 관한 연구 (1) 1. 미세조작된 배의 생존성과 분리된 단일할구의 체외 발생태에 관한 연구

박수봉,최광수,전익수 경북대학교 유전공학연구소 1995 遺傳工學硏究所報 Vol.10 No.1

This study investigated survival and development in vitro of micromanipulated embryos and biopsied blastomeres during early development of mouse embryos. The micromanipulated 4-cell and intact 4-cell embryos in Medium 2 developed to blastocyst stage by 83.3% and 90.4%, respectively. Blastomeres either biopsied or separated of 4-cell embryos in vitro developed to trophoblastic vesicle by 80.8% and 83.3%, respectively. After transfer to pseudopregnant recipient mice, the offspring races of biopsied embryos and intact embryos were 36% and 48.6%, respectively. The results suggest that the biopsy of single blastomeres from 4-cell mouse embryos have no detrimental effect on survival of embryos and blastomeres.

미세현미조작된 생쥐배의 발생과 단일할구의 성감별 기술에 관한 연구 (1) 2. 미세조작에 의해 분리된 할구의 염색체 분석조건에 관한 연구

박수봉,최광수,전익수,정범식 경북대학교 유전공학연구소 1995 遺傳工學硏究所報 Vol.10 No.1

The study was carried out to investigate the optimum condition of C-handing for sexing mouse embryo by chromosome analysis of single blastomere. The sexing rate was 42.9% with the 114 blastocyst embryos which were used C-handing after being hypotonic-treated in 1% sodium citrate for 30 minutes. The separated blastomeres for 4-cell embryos were used For C-banding after being hypotonic-treated in 1% sodium citrate for 20-30, 10-15 and 1-2 min., and the sexing rate For different treatment times were 5.5%, 0% and 0%, respectively. The separated blastomeres from 4-cell embryos were used for C-banding after being hypotonic-created in 30% fetal calf serum for 20-30. 10-15 and 1-2 min., and the sexing rate for different treatment tunes were 18.5%. 0% and 0%, respectively.