초록 ▼

Ⅳ. 연구개발결과
○ 공여세포주에 acteoside를 처리하여 세포주기 동기화 확인, 체세포 복제에 사용하여 배 발달율 향상 확인
○ acteoside 처리한 공여세포주의 세포사멸과 활성산소종 감소 확인, 이를 사용하여 복제견 1두 생산
○ 난자 제공견에 PMSG와 hCG를 주사하여 과배란처리 유도한 결과 일반견보다 회수되는 난자 수 약 3배 증가
○ 체내 미성숙 난자 회수시 체외성숙 배양하여 약 67%의 성숙률 확인
○ 개 양수 유래 중간엽 줄기세포의 신경 분화능 확인
○ 개 유래 중간엽 줄기세포와

Ⅳ. 연구개발결과
○ 공여세포주에 acteoside를 처리하여 세포주기 동기화 확인, 체세포 복제에 사용하여 배 발달율 향상 확인
○ acteoside 처리한 공여세포주의 세포사멸과 활성산소종 감소 확인, 이를 사용하여 복제견 1두 생산
○ 난자 제공견에 PMSG와 hCG를 주사하여 과배란처리 유도한 결과 일반견보다 회수되는 난자 수 약 3배 증가
○ 체내 미성숙 난자 회수시 체외성숙 배양하여 약 67%의 성숙률 확인
○ 개 양수 유래 중간엽 줄기세포의 신경 분화능 확인
○ 개 유래 중간엽 줄기세포와 태아 섬유아세포의 공여세포 활용성 탐색
○ 티타늄 바늘을 개발하여 세포-난자 융합율을 안정화시킴
○ 1협동과제에서 공급받은 특수목적견 체세포를 이용하여 복제견 3두 생산.
○ 개 골수유래 중간엽 줄기세포주의 구축
○ 개 혈액유래 중간엽 줄기세포주의 구축 및 이를 이용한 복제견 3두 생산
○ 활성화 조건, 수정란 배양액에 따른 개-돼지 이종간 복제수정란 생산연구
○ 개 체세포에 대한 히스톤 탈아세틸화 억제제인 Sciptaid와 히스톤 아세틸화 활성화제인 CTB의 효과에 대한 연구
○ Sciptaid 처리된 개 복제수정란 이식시, 분만율과 산자 생산율에 있어서 대조군과 차이가 없음
○ 개 복제수정란에 대한 G1, G2를 이용하여 체외배양시 세계 최초로 배반포 생산
○ 개 복제를 위한 핵치환 공여세포주로 사용될 유도만능줄기세포 생산 시스템 구축
○ 이종간 체세포핵치환 방법을 이용하여 개 복제를 위한 핵치환 공여세포주로 사용될 배아줄기세포의 생산 가능성 조사
○ 개 유도만능줄기세포의 최적 배양 조건 확립
○ 개 유도만능줄기세포에 대한 viramin C 처리시 효율 증진, 만능성, 분화능 검증

Abstract ▼

Somatic cell nuclear transfer (SCNT) is a well-known laboratory technique. The principle of the SCNT is reprogramming a somatic nucleus to pluripotent state by injecting a somatic cell into a recipient oocyte from which its own nucleus has been removed. For that reason the nucleus donor cells are co

Somatic cell nuclear transfer (SCNT) is a well-known laboratory technique. The principle of the SCNT is reprogramming a somatic nucleus to pluripotent state by injecting a somatic cell into a recipient oocyte from which its own nucleus has been removed. For that reason the nucleus donor cells are considered as a crucial factor in SCNT. Synchronization of cell cycle at G0/G1 stage of the nucleus donor cells can be induced by contact inhibition or serum starvation. Here acteoside, a phenylpropanoid glycoside compound, was investigated to determine whether it is applicable for induction of cell cycle synchronization, cytoprotection and SCNT efficiency in canine fetal fibroblasts. Primary canine fetal fibroblasts were treated with acteoside (10, 30, 50 μM) for various duration (24, 48 and 72 hours). No significant difference was found among acteoside, contact inhibition and serum starvation regarding cell cycle synchronization at G0/G1 stage. However, of these three treatments, serum starvation resulted in significantly high level of reactive oxygen species (ROS) (99.50 ± 0.3%) and apoptosis. The results revealed that acteoside reduced ROS and apoptosis activities including necrosis in canine fetal fibroblasts, and influenced beneficial effects on cell survival. These data suggested that canine fetal fibroblasts treated with acteoside were successfully arrested at the G0/G1 stage. Moreover, the reconstructed embryos with nucleus donor cells after acteoside treatment produced a healthy cloned dog, but not the embryos with nucleus donor cells after contact inhibition. In conclusion, acteoside induced cell cycle synchronization of nucleus donor cells would be an alternative method to improve the efficiency of canine SCNT due to its cytoprotective effects.
Histone methylation, acetylation and DNA methylation are the most important factors in somatic cell nuclear transfer (SCNT). Various chemicals have been used to evaluate the SCNT efficiency in mammals, such as treatment with a histone deacetylase inhibitor (HDACi) and a DNA methyltransferase inhibitor (DNMTi). Especially, histone deacetylase inhibitors, such as trichostatin A (TSA), 6-(1,3-dioxo-1H, 3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide (Scriptaid), valproic acid (VPA), sodium butylate, suberoylanilide hydroxamic acid (SAHA), m-carboxycinnamic acid bishydroxamide (CBHA) and oxamflatin have been used to improve embryonic development in various species. But, to our knowledge, there is no study in case of canine. Therefore, this study aimed to evaluate the effect of scriptaid treatment on canine ear fibroblast and early development of canine cloned embryos produced from canine ear fibroblast pre-treated with a scriptaid. Scriptaid was directly treated in canine fibroblast (≥80% confluency) growth media for 24h, and pre-treated cells were used for analysis. To evaluate the toxicity on cEF, we used six concentration of scriptaid (0, 100, 250, 500, 750 and 1000 nM/ml) for TUNEL assay. There was no difference among treatment concentration.
Histone acetylation (H3K9, H3K14 and H4K5) revels significantly increased in 500 nM/ml treatment group. The expression of acetyltransferase (GCN4 and HAT1), deacetylase (HDAC6) and anti-apoptotic gene (Bcl2) was significantly decreased in 500 nM/ml concentration, especially H3K14 acetylastion was the most affective. So, we fixed concentration (500 nM/mL) for further studies. Also, In SCNT embyos from the canine ear fibroblast pre-treated with scriptaid, H3K14 acetylation levels were significantly increased in one cell and two cell stages when compared with control group. These result demonstrated that scriptaid can affect canine somatic cell nuclear reprogramming.
The dog has been regarded as a good human disease model for a number of reasons, especially with respect to hereditary diseases for their physiological and biochemical similarities with human. Thus, the establishment of canine pluripotent stem cells will greatly facilitate the use of the dog in the development of stem cell-based therapies and regenerative medicine. And also, previous reports described that using pluripotent stem cells as a donor for nuclear transfer could increase their development efficiency and decrease errors that arise during reprogramming process. It means successful generation of dog pluripotent stem cell might help production of cloned animal. However, although a lot of efforts made to develop canine embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), success has been elusive for their unknown in vitro culture condition.
In this experiments, iPSCs were successfully derived from canine fetal fibroblasts by retroviral OSKM transduction. These iPSCs have a morphology resembling previously described canine ESCs and each cell had a high nuclear to cytoplasmic ratio. Human four factors were successively integrated into canine genome and expressed. After 3 passages, those transgenes were silenced which means their successful reprogramming. They were positive for alkaline phosphatase and expressed pluripotency markers such as OCT4, SOX2, NANOG and TRA-1-60. Pluripotent gene expression such as Oct4, Nanog, Rex1 and Telomerase also revealed that the cells were successfully reprogrammed. In addition, embryoid body formation from these cells and their differentiation confirmed their differentiation potential. They could maintain their morphology and pluripotency under human stem cell culture media supplemented with LIF, bFGF, GSK3 β inhibitor and MEK inhibitor. In addition, under valproic acid or vitamin C treatment, cells were reprogrammed more efficiently. Therefore, these results suggest that human four transcription factors could reprogram canine somatic cells into pluripotent state. Furthermore, optimal culture condition confirmed in this experiment could be applied successful canine ESCs derivation.