메추리 수정란의 대리난각 배양

전익수,최광수,손시환,오노타마오 ( I . S . Jeon,K . S . Choi,S . H . Sohn,T . Ono ) 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.

배반엽 세포 주입에 의한 닭 - 메추리 키메라 생산

전익수,최광수,손시환,오노타마오 ( I . S . Jeon,K . S . Choi,S . H . Sohn,T . Ono ) 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.

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.

닭 - 메추리 속간잡종의 특성과 핵형 연구

전익수,정일정,최광수,손시환 ( I . S . Jeon,I . C . Cheong,K . S . Choi,S . H . Sohn ) 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.

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.

ICSI 프로그램에서 생쥐 투명대를 이용한 고환조직내 정자의 동결

서태광,전병균,류은경,이은숙,류재웅,손시환,문진수,김광철,Suh, Tae-Kwang,Jeon, Byeong-Gyun,Ryu, Eun-Kyung,Lee, Eun-Sook,Ryoo, Zae-Yoong,Sohn, Sea-Hwan,Moon, Jin-Soo,Kim, Kwang-Chull 대한생식의학회 1997 Clinical and Experimental Reproductive Medicine Vol.24 No.2

The survival rate and motility recovered after cryopreservation of testicular spermatozoa in testicular sperm extraction (TESE)-ICSI program is low. The purpose of this study was to assess the availability and efficiency of mouse empty zona pellucida in cryopreserving human TESE spermatozoa. Mouse empty zonae pellucidae were obtained by extraction of cytoplasm with or without cytochalasin B treatment. Motile sperm from proven-fertile donor and two azoospermic patients after TESE were individually inserted into empty zona pellucida and cryopreserved. Two to five days after cyropreservation, the frozen sperm were thawed and the rates of recovery and motility were observed. The ooplasmic extraction rates of control (N=80) and cytochalasin B treated oocytes (N=80) were 94.0% and 96.2%, respectively (p>0.05). The post-thaw recovery rates of spermatozoa and rates of motility recovery of ejaculate (N=70) and testicular (N=70) sperm were 97.1%, 97.1% and 95.7%, 94.3%, respectively (p>0.05). The results of this study showed that the mouse zone pellucida is useful for cryostorage of single testicular spermatozoa.

유전 및 육종 : 혈액세포의 텔로미어 함량을 이용한 소의 연령예측

김현섭 ( Hyun Sub Kim ),최창용 ( Chang Yong Choe ),전광주 ( Gwang Joo Jeon ),손시환 ( Sea Hwan Sohn ),최나은 ( Na Eun Choi ) 한국동물자원과학회 ( 구 한국축산학회 ) 2010 한국축산학회지 Vol.52 No.5

텔로미어란 진핵세포의 염색체 양 말단에 있는 DNA-단백질 복합체로서, 특정단백질과 TTAGGG의 반복염기서열로 구성되어있다. 이들의 기능은 핵 내 염색체의 안정성에 본질적으로 작용함으로 세포의 노화와 직접적 관련이 있다고 알려져 있다. 본 연구에서는 소의 간기상태의 백혈구 세포를 대상으로 연령별, 품종별, 성별간 telomeric DNA 함량을 분석하여 이러한 요인들이 텔로미어 함량에 미치는 영향을 살펴보고 또한, 텔로미어 함량을 이용한 개체의 연령예측 가능성을 제시하고자 하였다. 소의 텔로미어의 함량 분석은 l개월령에서 166개월령의 한우 및 홀스타인종 460두를 대상으로 telomeric DNA probe를 이용한 Q-FISH 방법으로 분석하였다. 분석 결과 소에 있어서 연령이 증가함에 따라 telomeric DNA 함유율이 일관되게 점진적으로 감소되는 양상을 보였다. 소의 품종간 telomeric DNA 함유율을 비교한 결과 한우의 telomeric DNA 함량이 홀스타인종에 비해 유의적으로 높게 나타났으며, 성별 간에도 수컷이 암컷에 비해 유의적으로 높은 telomeric DNA 함유율을 나타내어 품종별, 성별 모두 텔로미어 함유율의 유의적인 차이가 있음 확인 할 수 있었다(P<0.01). 따라서 요인별 유의적 차이가 있음으로 한우 암컷 및 홀스타인 암컷에 대한 각기 연령예측 회귀함수를 추정하였다. Telomeric DNA 함량을 독립변수(X)로 하고, 연(월)령을 종속변수(Y)로 설정하여 2차회귀식을 도출한 바 한우 암컷의 경우 Y=38.102X2-220.103X+318.309(P<0.0001, R2=0.8019)이고, 홀스타인 암컷은 Y=42.799X2-199.682X+242.106(P<0.0001, R2=0.8379)으로 분석되었다. 이상의 두 회귀식 모두 유의한 함수로 결정계수(R2) 또한 0.8 이상의 높은 상관 값을 보임에 따라 본 회귀식으로 소의 연령 예측이 가능함을 제시하고자 한다. Telomeres at the end of chromosomes consist of tandem repeats of (TT AGGG)n DNA sequence and associated proteins. Telomeres have the essential functions in chromosome stability and genome integrity and are hence related to cell senescence and cancer. This study was carried out to quantify the amount of telomeric DNA and establish age prediction equations by using the quantity of telomeric DNA for cattle. Analysis of the telomere quantity of the lymphocytes was performed at different age, across breeds and between different sexes of cattle. We quantified the amount of telomeric DNA by the Q-FISH technique using the telomeric DNA probe in 460 cattle at age of 1-166 months in Korean Cattle and Holstein breeds. In results, we found that the amount of telomeric DNA decreased gradually with age. The amount of telomeric DNA of Korean Cattle was significantly higher than that of Holstein breed (P<0.01). In addition, the amount of telomeric DNA in male was significantly higher than that in female (P<0.01). Using the relationship between age and the amount of telomeric DNA in cattle, age predicting equations were established as a result of regression analysis. Because sex and breeds influenced telomeric DNA quantity, the age prediction equations were estimated separately in Korean Cattle females and Holstein females. The regression equations were Y = 38.102X2 - 220.103X + 318.309 (P<0.0001, R2=0.8019) in Korean Cattle females and Y = 42.799X2 - 199.682X + 242.106 (P<0.0001, R2 = 0.8379) in Holstein females, where the X was quantity of telomeric DNA and Y was predicted age in months. These equations predicted the age of cattle with high significance and accuracy and have high R square values. Thus, it could be possible to scientifically predict the age using the above equations for Korean Cattle and Holstein females.

Telomere Length and Telomerase Activity of Korean Native Chicken

D. S. Choi,M. J. Lee,J. A. Park,Y. J. Kim,C. Park,I. S. Jeon,S. H. Sohn(손시환) 한국가금학회 2006 한국가금학회 정기총회 및 학술발표회 Vol.23 No.-

The telomere length and telomerase activity of Korean Native Chicken cells were analyzed along with aging. The cells were collected from brain, heart, liver, kidney and testis tissues during physiological stages. Telomere length was analyzed by Q-FISH using the telomeric DNA probe. Telomerase activity was performed by TRAP assay. The amount of telomeres on chicken cells was decreased along with aging in most tissues. Furthermore, telomere quantity was significantly different among tissues. The telomeres in gonadal cells had much more than those of other cells. The telomerase activity was down-regulated in cells of brain, heart and liver tissues. Whereas gonadal cells showed a constitutive telomerase activity 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.