• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.70-70
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• 2002

형질전환 가축을 생산하기 위하여 최근 체세포 복제 기법을 이용하고 있다. 이러한 체세포를 이용한 형질전환 동물의 생산에는 체세포내에 유전자의 도입 효율이 직접적인 영향을 주게 된다. 따라서 본 연구는 세포내 유전자의 transfection 효율을 높이고자 한우의 체세포를 이용하여 여러 가지 조건에서 유전자 도입을 실시하였다. 세포내 유전자 도입 방법은 electroporation (EP) 방법을 이용하였다. 사용한 세포는 소의 귀세포(KbESF), 태아섬유아세포 (KbFF), 그리고 대조구로서 CHO cell을 이용하여 GFP 유전자를 도입하였다. EP는 0.4 cm cuvette을 사용하였고, voltage는 0.25 kV, 그리고 field strength 는 0.625 kV/cm 조건으로 실시하였으며, pulse times은 각각 1, 2, 또는 3회를 사용하였다. KbFF와 KbESF에서는 각각 pulse times을 증가시킬수록 유전자도입 세포수가 증가하였으나 (KbFF: 81, 634, 1,065 cells/$10^{6}$ cells, KbESF: 1,011, 5,567, 15,408 cells/$10^{6}$ cells), CHO cell에서는 pulse times을 증가시킬 수록 오히려 유전자도입 세포수가 감소하였다 (CHO: 1,591, 687, 297 cells/$10^{6}$ cells). 그리고 2주 동안 neo selection을 실시 한 결과 KbFF, KbESF, CHO에서 각각 93, 35, 184 colony가 선발되었으며, 이 중 65.6%, 8.6%, 4.3% 가 GFP 형광 발현 colony로 나타났다. 한편 CHO cell에서 transfection cell수가 감소된 것은 EP의 자극으로 인해 손상된 세포가 많이 발생한 것으로 나타났다. 또한 neo selection에서 선발된 colony중 GFP가 발현되지 않거나 일부만 발현되는 colony들이 많이 발생하였는데, 이것은 세포내 유전자가 transfection되지 않은 세포도 neo selection에서 선발된다는 것을 제시하고 있다. 따라서 체세포를 이용한 형질전환동물 생산을 위해서는 세포내 유전자 도입과 선발 과정에서 나타난 colony에 대하여 보다 엄격한 screen을 하는 것이 필요한 것으로 생각된다.

• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.53-58
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• 2006

The study was conducted to evaluate the effects of culture period and fusion method on the development of somatic cell nuclear transfer (SCNT) embryos reconstituted with Korean bovine fetal fibroblast cells (KbFF) and Korean bovine adult ear skin fibroblast cells (KbESF). KbFF were isolated from a day 51 Korean cattle (Hanwoo) fetus, and KbESF were isolated from a 28 month old Hanwoo calf. The cells were cultured up to 15 weeks (passage 15) in vitro for SCNT. Chamber and electrode needles were used for comparing fusion of reconstituted eggs. The doubling times of KbFF and KbESF were 17.3 hr and 24.3 hr, respectively. The fusion and cleavage rates were significantly higher in needle group (76.1 and 81.2% respectively, P<0.05) than those in chamber group. However, the blastocyst development rate was not different between both groups. Fusion and cleavage rates of NT eggs reconstituted with KbESF did not affected by passage number, however, blastocyst rates were lower in passage $1{\sim}4$ group (21.3%) than passage $5{\sim}8$ (39.4%) and $13{\sim}15$ groups (40.4%, P<0.05). Whereas, fusion rate was lower in passage $1{\sim}4$ group (61.5%) than those of passage $5{\sim}8$(75.0%) and $13{\sim}15$ (76.8%) groups, but cleavage and blastocyst rates were similar regardless of passage number in the KbFF. The results suggest that fusion method can affect the development of SCNT embryos, whereas the long term culture up to 15 passages may not affect the development of SCNT embryos.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.42-42
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• 2003

복제기술은 기존의 형질전환 동물 생산의 효율을 향상시킬 수 있는 기술로서 인정하고 있으며 또한 이를 이용하여 형질전환 동물의 생산이 이루어지고 있다. 따라서 본 연구는 표지유전자 (GFP)와 유용유전자 (hFSH)를 이용하여 임신 45일령에 채취한 태아섬유아세포에 transfection 하고, transfection 된 세포의 효율적인 선발과 이를 이용한 형질전환 복제 수정란을 생산하고자 실시하였다. 대조구 (KbFF), GFP (79KbFF-GFP c-3) 및 hFSH (79KbFF-hFSH n-1)에 공시한 세포는 모두 동일한 태아유래의 세포 (모 79, 부 KPN178,♂)를 이용하였다. pAB-eGFP와 hFSH 유전자는 각파 electroporation 방법을 이용하여 transfection 하고, 이를 2주 동안 G418로 배양하며 selection 하였다.

• The Korean Journal of Mycology
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• v.26 no.4 s.87
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• pp.511-518
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• 1998

Anti-complementary assay for immuno-stimulating polysaccharide screening, human tumor colony-forming assay for discovering anti-tumor drugs, and toxic assay against mouse were performed to examine pharmacological activities of polysaccharides extracted from fruiting bodies of Jang-soo mushroom (Fomitella fraxinea). Hot water $(100^{\circ}C,\;FF-I)$, 1% ammonium oxalate solution $(80^{\circ}C,\;FF-II)$, and 5% sodium hydroxide solution $(80^{\circ}C,\;FF-III)$ were used for extraction of three polysaccharides from fruiting bodies of it. Anti-complementary activity of FF-I was more effective than the others. FF-I was further fractionated into three groups of polysaccharide by DEAE-Sephadex A25 column chromatography (FF-NP, FF-AP1, and FF-AP2). FF-AP1 showed not only the highest anti-complementary activity but also the growth-inhibitory activity against Snu-I (human stomach cancer cell) among 9 kinds of human tumor cell lines. But FF-AP2 exhibited its activity against Hep-2(larynx cancer) and KB(mouth epidermal cancer) cell lines at $500\;{\mu}g/ml$ although its anti-complementary activity was lower than it of FF-AP1. When FF-I was orally administrated to mice with dosage of 5000 mg/kg, no remarkable changes were observed in viewpoint of tissue-pathology.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.220-220
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• 2004

The purpose of this study was to development of transgenic cow using the nuclear transfer. To secrete hFSH in urea, the vector was constructed with UPII promoter. The fetal fibroblast cells (KbFF) were constructed from pregnant day 45 male fetus. The hFSH genes were cotransfected with pcDNA3 (neo) vector to KbFF cells by electroporation. (omitted)

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.34-34
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• 2002

핵이식 방법은 형질전환 동물을 생산하기 위한 여러 가지 방법 중 최근에 많이 이용되고 있다. 본 실험은 형광단백질 유전자 (green fluorescence protein, GFP)가 도입된 태아섬유아세포를 이용 핵이식을 하여 형질전환 수정란의 생산효율을 검토하기 위하여 실시하였다. GFP 유전자는 임신 45-55 일령의 태아섬유아세포 (KbFF3)에 electroporation방법으로 transfection을 실시하였다. (중략)

• Journal of Embryo Transfer
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• v.21 no.1
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• pp.13-20
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• 2006

The purpose of this study was to develope the transgenic cattle expressing hFSH into the urine using the nuclear transfer. To produce the interest gene in urine, the specific vector was ligated with hFSH gene undo. maUII promoter. The fetal fibroblast cells (KbFF) were isolated from a 45-day male fetus. The hFSH gene was co-transfected with pcDNA3 (neo) vector to KbFF cells by electroporation. The gene-transfected cells were cultured with G-418 selection medium for 2 weeks. Selected colonies were confirmed by PCR. For nuclear transfer, enucleated bovine oocytes were transferred with hFSH transfected or nontransfected fetal fibroblasts. The cleavage and blastocyst formation rates were significantly lower (p<0.05) in cloned embryos transfected with hFSH gene (68.7% and 15.7%) than in those non-transfected (67.6% and 24.5 %), respectively. Apoptosis analysis showed no difference between hFSH transfected and non-transfected blastocysts (p>0.05). The blastocysts were transfected to 77 (control 24, hFSH 53) recipient cows. Two calves were born (1.9%) following transfer with NT embryos transfected with hFSH gene, but they were confirmed not to be transgenic calves. This result shows that the hFSH colonies were mixed with transfected and non transfected cells. Further research will be needed for selection and establishment of gene transfected cells.

• Journal of Microbiology and Biotechnology
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• v.16 no.2
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• pp.286-290
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• 2006

A genomic library of Streptococcus vestibularis ATCC49124 was constructed in an E. coli plasmid vector, and the urease-positive transformants harboring the urease gene cluster were isolated on Christensen-urea agar plates. The minimal DNA region required for urease activity was located in a 5.6 kb DNA fragment, and a DNA sequence analysis revealed the presence of a partial ureI gene and seven complete open reading frames, corresponding to ureA, B, C, E, F, G, and D, respectively. The nucleotide sequence over the entire ure gene cluster and 3'-end flanking region of S. vestibularis was up to 95% identical to that of S. salivarius, another closely related oral bacterium, and S. thermophilus, isolated from dairy products. The predicted amino acid sequences for the structural peptides were 98-100% identical to the corresponding peptides in S. salivarius and S. thermophilus, respectively, whereas those for the accessory proteins were 96-100% identical. The recombinant E. coli strain containing the S. vestibularis ure gene cluster expressed a high level of the functional urease holoenzyme when grown in a medium supplemented with 1 mM nickel chloride. The enzyme was purified over 49-fold by using DEAE-Sepharose FF, Superdex HR 200, and Mono-Q HR 5/5 column chromatography. The specific activity of the purified enzyme was 2,019 U/mg, and the Michaelis constant ($K_{m}$) of the enzyme was estimated to be 1.4 mM urea. A Superose 6HR gel filtration chromatography study demonstrated that the native molecular weight was about 196 kDa.