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Wee G.,B. H Sohn,J. S. Park,D. B. Koo,K. K. Lee,Y. M. Han 한국동물생명공학회(구 한국동물번식학회) 2003 Reproductive & developmental biology Vol.27 No.1
인체 트롬보포이에틴(hTPO)은 megakaryopoiesis 과정에 주요한 역할을 하는 사이토카인이다. 따라서 이러한 트롬보포이에틴을 유선조직에서 직접적으로 발현시키기 위하여 소 베타 카제인 프로모터, 인체 트롬보포이에틴 cDNA 및 네오유전자로 구성된 발현벡터를 구축하였다. 소 귀조직 세포로부터 유도된 섬유아세포에 lipoffctamine을 이용하여 발현벡터(pBT-L n대)의 삽입을 유도하였다. G4l8 저항성을 지닌 세포의 콜로니 형성을 유도하기 위하여 2주 이상 배양을 실시하였다. 형질전환 콜로니는 PCR에 의해 동정하였으며, 이들 콜로니를 핵치환 전까지 계속적으로 증식을 유도하였다. 형질전환 세포에 의해 재구성된 난자는 전기적인 융합과 calcium ionophore와 6-DMAP를 이용한 활성화를 실시하였으며, 체외에서 7일간 배양을 실시하였다. 총 35개의 콜로니를 PCR에 의해 분석한 결과, 이 중 29(82.9%)개가 형질전환된 콜로니였다. 형질전환된 세포로 재구성된 난자의 난할율 및 배반포로의 발달율은 65.1%와 23.8%로 나타났다. 형질전환된 세포로 재구성된 난자로부터 발달한 29개의 배반포 중 27개가 형질전환으로 확인되었다. 따라서 이러한 결과들은 형질전환 소 수정란을 형질전환된 세포를 이용한 체세포 복제 기법을 통해 효과적으로 생산할 수 있다는 것을 제시하고있다. Human thrombopoietin (hTPO) is a cytokine that plays a central role in megakaryopoiesis. To direct hTPO expression in the mammary gland, an expression vector was constructed by combining the promoter of bovine beta-casein gene, cDNA of hTPO and neomycin resistance gene (pBT-L neo). Fibroblast cells derived from cow′s ear skin tissue were transfected with the expression vector (pBT-L neo) using Lipofectamine. Transfected cells resistant to G418 trea?nt were cultured to form the colonies for more than 2 weeks. The transformed colonies identified by PCR were further expanded prior to nuclear transfer. Reconstructed oocytes with transformed cells were electrofused, activated using calcium ionophore and 6-DMAP, and cultured in vitro for 7 days. Of 35 cell colonies analyzed by PCR, 29 colonies (82.9%) were positive for the hTPO gene. Cleavage and developmental rates to the blastocyst stage of reconstructed embryos with the transformed cells were 65.1% and 23.8%, respectively Of 29 blastocysts that developed from reconstructed embryos with the transformed cells, 27 embryos (93.1%) were transgenic. These results indicate that transgenic bovine embryos can be efficiently produced by somatic cell nuclear transfer using transformed cells.
효율적인 돼지 복제수정란 생산에 관한 연구 : Ⅱ.탈핵 여건의 확립
위갑인,강만종,문승주 충남대학교 형질전환복제돼지연구센터 2004 논문집 Vol. No.8
This study was carried out to improve of enucleation effciency on porcine recipient oocytes preactivated. In ethanol or Ca^(2+) ionophore, effect of repeating and combinational activation with 6-DMAP or cycloheximide compared with alone activated treatment. Recipient oocytes's activation by Ca^(2+)ionophore combined with 6-DMAP or cycloheximide were significantly higher than alone treatment(P<O.05). Between repeating and alone treatments were not significantly different. In ethanol, repeating treatment was significantly lower than alone(P<O.05), and combination treatments were not significantly different. On the basis of these results, efficiency of enucleation, electrical fusion and in vitro development compared preactivated with non-preactivated recipient oocytes. Enucleation and fusion rates of preactivated oocytes were improved significantly compared with non-preactivated oocytes(90.7%, 71.8 vs 77.8%, 61.1%; P<O.05). Behind the back, cleavage and in vitro development rates were significantly lower than nonpreactivated oocytes(38.7%, 19.3% vs 68.8%, 30.6%; P<0.05).
Wee, S.B.,An, G.S.,Han, J.S.,Oh, H.C.,Choi, S.C. Ceramurgica ; Elsevier Science Ltd 2016 CERAMICS INTERNATIONAL Vol.42 No.4
The co-dispersion behavior and interactions of nano-ZrB<SUB>2</SUB> and nano-SiC in a non-aqueous solvent were investigated. Polyvinylidene fluoride (PVDF) and oleic acid were adopted as dispersants to study the stable co-dispersion mechanism in a non-aqueous solvent. Because N-methyl-2-pyrrolidone (NMP) has a relatively high dielectric constant among non-aqueous solvents, NMP was introduced; NMP was found to exhibit a good affinity with dispersants. ZrB<SUB>2</SUB> and SiC were mixed in NMP with or without dispersants. ZrB<SUB>2</SUB> and SiC in a paste without dispersants aggregated due to the poor repulsion force by a double-layer overlapping. The paste with PVDF was more homogeneous than the paste with oleic acid. The steric hindrance effect of PVDF was evaluated using SEM image analysis. FT-IR analysis results show that PVDF and oleic acid were chemically adsorbed onto the particle surface.