• Asian-Australasian Journal of Animal Sciences
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• 제17권6호
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• pp.743-749
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• 2004

The gene encoding human serum albumin (HSA) was cloned from human liver cDNA library by PCR. The HSA cDNA in size of 2,176 bp, including 1,830 bp of open reading frame, was cloned into the plasmid carried with the 5'flanking sequence of goat $\beta$-casein gene (-4,044 to +2,025 bp) to get a tissue specific expression vector in mammary gland named pGB562/HSA (12.5 kb). A 9.6 kb DNA fragment in which the sequence is in order of goat $\beta$-casein gene regulatory sequence, HSA cDNA and SV40 polyadenylation signals was isolated from the pGB562/HSA by SacI and DraIII cutting, and used to microinject into the pronuclei of mouse fertilized eggs to produce transgenic mice. Three transgenic mice (2 female and 1 male) were identified by PCR and dot Southern blot analysis. The copy numbers of integrated transgene were more than 10 copies in line #21 and #26 as well as over 50 copies in line #31 of transgenic mice. HSA protein collected from the milk of lactating transgenic mice was confirmed by immuno-detection of Western and slot blot. The concentrations of HSA in the milk were from 0.05 to 0.4 mg/ml. An obvious antigen and antibody conjugate could be observed in immunohistochemical stain of mammary gland tissue from lactating day 11 of HSA transgenic mice. The transmission of transgene and its expression was recognized according to the results of RT-PCR and sequences analyses of their progeny.

• 한국식품저장유통학회지
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• 제16권5호
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• pp.782-788
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• 2009

감마선 조사 계란의 유전독성학적 안전성을 평가하기 위하여Escherchia coli PQ37 균주를 이용한 SOS chromotest, Chinese hamster lung cell을 이용한 염색체 이상 시험의 in vivo 시험과in vivo 시험으로 마우스를 이용한 소핵시험을 실시하였다. 대사활성계 적용 및 미적용의 in vitro의 모든 시험에서 음성대조군과 통계학적으로 유의적인 차이는 없었다. 또한in vivo 시험에서도 시험물질을 투여한 모든 투여군에서 음성대조군에 비해 통계학적으로 유의적인 독성을 나타내지 않았다. 따라서 감마선 조사된 난백 및 난황은 본 시험조건하에서 직접변이원이나 간접변이원으로 작용하지 않는 것으로 사료된다.

• Parasites, Hosts and Diseases
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• 제29권4호
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• pp.371-380
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• 1991

간흡충 피낭유충의 경구감염 또는 간흡충 성충의 대사산물의 피내주사로 감작한 근교계 nude 및 DS 마우스의 복강삼출세포와 혈청이 간흡충 감염에 대한 면역을 동승(syngeneic) 마우스에 이입하는지 여부를 구명하기 위해 대조군 마우스에서의 EpG, 부하충체수 및 모장당 plaque 형성세포수를 기준으로 감작군에서의 그 성적과 비교하였다. Challenge 감염후 EpG의 변동은 감작군과 비감작 대조군 마우스와 nude 및 DS 마우스와의 사이에 유의한 차를 인정할 수 없었다. 간흡충 성충의 회수률에서는 감작군과 비감작 대조군 사이에 유의적 차를 인정할 수 없었으나 nude 마우스는 DS 마우스에 비하여 다소 높은 회수률을 나타내었다. 비장에서 plaque 형성세포는 복강 삼출세포 $5{\times}10^5$와 Group II DS 마우스의 혈청 0.1 ml를 복강에 감작한 제4군에서만 소수 검출할 수 있었다. 이상의 성적으로 미루어 보아 세포성 면역계에 결손이 있다고 알려진 근교계 nude 및 DS의 복강삼출세포와 혈청에 의해서는 간흡충에 대한 면역이 이전되지 않음을 확인하였다.

• 한국가금학회:학술대회논문집
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• 한국가금학회 2002년도 가을 학술발표논문집
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• pp.96-97
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• 2002

Primordial germ cells (PGCs) are the progenitors of the sperms or eggs of adult. Evidence suggests that the specification of primordial germ cells (PGCs) in the mammalian embryo does not depend on maternal determinants. Recent previous studies in the mouse has shown that several bone morphogenetic proteins (BMPs) are required for the formation of PGCs. However, there is no study about the effect of BMPs on avian PGCs. Here, we studied the effects of recombinant human BMP-2 (rhBMP-2) and recombinant human BMP-4 (rhBMP-4) on chicken blastodermal cells in culture. As a results, the addition of rhBMP-2 and rhBMP-4 increased the number of SSEA-1 positive cells in dose-dependent manner. However, there is no synergic effect by using both rhBMP-2 and rhBMP-4.

• Clinical and Experimental Reproductive Medicine
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• 제26권2호
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• pp.171-178
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• 1999

The follicular fluid (FF) of ovary contains various biological active products which affected on the growth of follicles and the fertilization of oocyte in physiological reproductive process of mammals. This study was designed to determine the effects of human FF on fertilization of oocyte and embryonal development in vitro culture. The FF was prepared as clear without blood contamination by needle aspiration from mature follicles of human at the time of oocytes retrieval for in vitro fertilization (IVF). As the medium for culture in vitro of embryonal cells, human tubal fluid (HTF) supplemented with follicular fluids at concentrations of 10%, 40% and pure FF were used. These effects were compared to control group of cultured embryos in HTF supplemented with 0.4% BSA (bovine serum albumin). For IVF, 64 eggs in control group, 67 eggs in 10% FF, 57 eggs in 40% FF and 64 eggs in pure FF were respectively allocated. And the rates of fertilization were almost similar in all groups as resulting 82.81% in control, 85.07% in 10% FF, 87.71% in 40% FF and 81.25% in pure FF. On the examination for embryonal cleavage from fertilized eggs, the rates of developing to 4 cell stage was similar in all groups, as results 98.11% in control, 98.27% in 10% FF and 98% in 40% FF but 78.84% in pure FF. And the rates of developing to 8-16 cell stage were significantly reduced as 44% in 40% FF and 44.23% in pure FF (p<0.05) compare to 71.69% in control media. As likewise, the rates of developing to morular stage were also significantly reduced to 36% (p<0.05) and 21.15% (p<0.01) respectively in 40% FF and pure FF. And the rates to blastocystic stage of embryo was lowest as 7.69% in pure FF (Table 1). The quality of embryonal cells on cleavage to the 8-16 cell stage was poorer, higher concentrations of FF. The rates of grade 1 in pure FF, as 23.07%, was lowest compare to those of other groups, in which the rates of grade 1 in control, 10% FF and 40% FF were 58.49%, 47.36% and 34% respectively. And on the contrary, the rate of grade 4 in pure FF was highest as 23.07%, while those were 5.66% in control, 8.77% in 10% FF and 20% in 40% FF (Table 2). On the viability of embryos, the rate of embryonal cell death was more rise, at the higher concentrations as well as longer exposure in the follicular fluid. At 48 hours after in vitro culture of embryos, the rate of survival embryos in pure FF was markedly lowered as 44.23%, compare to that of control (p<0.05). But there was not significant difference between the rates of survival embryos in each group beside the pure FF, which the rates were 77.35% in control, 70.17% in 10% FF and 60% in 40% FF respectively. And at 72 hours after in vitro culture, the rates of survival embryos were also significantly dropped to 21.15% in pure and 36% in 40% at concentration of FF compare to 62.26% in control (p<0.05, p<0.01). Finally, the rate of embryonal death at 96 hours after in vitro culture was highest as 82.69% in pure FF among all groups which those were 35.84 in control, 56.14% in 10% FF and 64% in 40% FF respectively (Fig. 1, 2, 3). In conclusion, this study suggests that the FF has no effects, in particular, to the in vitro fertilization of oocytes but exerted a bad effect to the cleavage, quality and viability of the embryonal cells during in vitro culture. However, the FF is harmful on embryonal development at conditions in higher concentration and especially on the embryos after $8{\sim}16$ cell stage.

• Journal of Yeungnam Medical Science
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• 제12권1호
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• pp.124-134
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• 1995

Ham's F-10 기본 배양액과 이 배양액에 여러가지 농도의 EDTA와 태아제대혈청을 단독 또는 같이 혼합한 배양액을 얻어 각각의 배양액에서 5-10주된 백서를 이용하여 2 세포기 배아의 단계적 분할정도를 96시간 동안 관찰하였다. Ham's F-10 기본 배양액에 비해 태아제대혈청이나 EDTA $50{\mu}M$에서 $100{\mu}M$을 단독 또는 함께 첨가한 배양액에서 2 세포기의 배아의 상실배기 난할률이 매우 높았으며 (p<0.05), 포배기 난할률은 태아제대혈청과 EDTA $50{\mu}M$에서 $100{\mu}M$을 첨가한 배양액에서 유의하게 높았다(p<0.05). 체외수정실에서 흔히 사용되는 태아제대혈청을 첨가한 배양액과 비교시 태아제대혈청과 EDTA $100{\mu}M$을 첨가한 배양액에서 상실기 난할률이 유의하게 높았다(p<0.05). 태아제대혈청과 여러 농도의 EDTA를 첨가한 배양액과 EDTA만 첨가한 배양액의 비교에서 태아제대혈청과 EDTA $50{\mu}M$ 및 $100{\mu}M$을 첨가한 배양액에서 EDTA $200{\mu}M$만 첨가한 배양액에 비해 상실기 난할률이 매우 유의하게 높았고(p<0.05), 포배기 난할률은 태아제대혈청과 EDTA $100{\mu}M$을 첨가한 배양액이 EDTA $200{\mu}M$을 단독 또는 태아제대혈청과 함께 넣은 배양액에 비해 유의하게 높았다(p<0.05). 결론적으로 Ham's F-10 기본 배양액에 태아제대혈청과 적정 농도의 EDTA $50{\mu}M$ 및 $100{\mu}M$을 첨가한 배양액에서 초기 수정란의 분할이 우수함을 알 수 있고, 이는 수정란의 배양시 적절한 배양액을 선택할 수 있는 기초 자료로서 유용할 것으로 사료된다.

• Parasites, Hosts and Diseases
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• 제34권1호
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• pp.21-26
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• 1996

우리 나라 고유의 인체 기생 장흡충의 하나인 참굴큰입홉충(Gvmophono천es seoi) 피낭유충에 대한 방사선 조사가 실험적 종숙주인 C3H 마우스 감염 능력에 어떠한 영향을 미치는지 관찰하였다. 유행지에서 굴을 채집하여 비조사 대조군 피낭유충 소사군 및 굴 조사군 등 3개 군으로 나누고, 각 군에서 얻은 피낭유충을 C3H 마우스 마리당 각각 100개씩 경구감염시킨 다음 7일째에 충체 회수율을 관찰하였다. 연구 결과, 피낭유충 조사군에서는 200 Gy 이상 조사시 대조군에 비해 유의하게 충체 회수율이 감소하였다. 생존 충체의 자궁 내 충란수는 50 Gy 이상 조사시 통계적으로 유의한 감소를 보였다. 굴 조사군에서는 50 Gy 이상 조사시 충체 회수율이 유의하게 감소하였으며 50 Gy 이상 조사시 자궁내 충란수의 유의한 감소를 보였다. 이상의 결과를 종합할 때 굴에 200-1 000 Gy를 조사할 경우 참굴큰입흡충 피낭유충의 갈멸 효과가 뚜렷하였으며, 따라서 이 흡충증 관리 방법의 하나로 이용할 수 있을 것으로 전망하였다.

• 한국가축번식학회지
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• 제15권1호
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• pp.67-77
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• 1991

本 試驗은 受精初期段階에서 생쥐卵子 透明帶의 機能을 糾明하고 種特異的인 精子受容體의 存在 및 構造理解를 위한 基礎硏究로서 ICR 생쥐의 透明帶를 免疫原으로 하여 토끼에서 抗透明帶抗體를 生産하고 免疫分析法 및 生物學的 檢査法으로 生産된 抗體가 생쥐 透明帶에 特異性이 있음을 確認한 후 이 抗體가생쥐 未受精卵의 體外受精에 미치는 影響과 또 이 抗體를 생쥐에 受動免疫하였을 때 受精에 미치는 影響을 調査하였던 바 다음과 같은 結果를 얻었다. 1. 處理區로서 抗血淸을, 對照區로서 對照血淸을 卵子培養液에 各各 0.3~10%로 含有한 培養液에서 생쥐의 未受精卵을 2時間 培養한 後 觀察하였을 때 對照區 卵子들의 透明帶가 正常的인 形態를 나타내었던 反面에, 處理區의 卵子들은 모두 透明帶 表面에 뚜렷한 沈澱層을 나타내었다. 이어 對照區와 處理區의 卵子를 0.1% pronase가 含有된 培養液으로 옮긴 후 觀察하였을 때, 對照區 卵子들의 透明帶가 5~7分만에 完全히 融解한 反面에, 處理區에서는 2~3時間까지 殘存하였다. 2. 處理區와 對照區 卵子를 FITC-goat anti-rabbit IgG가 含有된 培養液에서 養液한 후 螢光顯微鏡下에서 觀察하였을 때 對照區에서 對照血淸을 10% 含有한 培養液에서 培養한 일부 卵子들의 透明帶에서 희미한 螢光이 나타났으나 血淸稀釋度가 增加함에 따라 전혀 螢光을 나타내지 않았던 反面에, 處理區에서는 抗血淸이 1 : 1,000으로 含有된 培養液에서 培養한 卵子의 透明帶에서도 강한 螢光이 나타났다. 3. 帶抗體를 抗原으로 하여 間接酵素免疫分析法을 實施하였을 때 抗血淸과 對照血淸이 PBS에 1 : 200까지 稀釋되었을 때의 O.D.價는 별다른 差異를 나타내지 않았으나 血淸의 稀釋度가 增加함에 따라 O.D.價가 有意한 差異를 나타냄으로써 透明帶에 特異性을 갖는 抗體임을 確認할 수 있었다. 4. 抗血淸과 對照血淸이 各各 0.3~10%까지 含有된 培養液으로 생쥐의 未受精卵을 豫備培養한 다음 體外受精을 實施하였을 때의 受精率은 對照區에서 77.7%~87.7%이었던 反面에, 處理區에서는 0~29.8%로서 抗透明帶抗體가 생쥐卵子의 體外受精을 完全히 또는 顯著히 抑制하였다. 그러나 抗血淸과 對照血淸이 各各 2.5~10%까지 含有된 培養液으로 透明帶를 除去한 未受精卵의 體外受精을 하였을 때의 受精率은 處理區와 對照區에서 各各 93.0~98.3% 및 93.3~94.3%로서 受精抑制現象을 나타내지 않았다. 5. 抗血淸과 正常血淸, 그리고 兩 血淸으로부터 protein A-affinity chromatography에 의해 分離한 immunoglubulin G를 各各 생쥐에 受動免疫하였을 때, 對照區로서 對照血淸을 0.1~0.3ml 또는 control IgG를 1~3ml 投與했을 때의 各各의 受精率이 80.4~90.2%, 82.2~94.4%이었던 反面에, 處理區로서 抗血淸을 0.3ml 또는 immune IgG를 3mg 投與한 생쥐에서는 完全한 受精抑制現象이 確認되었다.

• 한국동물생명공학회지
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• 제34권3호
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• pp.148-156
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• 2019

The Transgenic livestock can be useful for the production of disease-resistant animals, pigs for xenotranplantation, animal bioreactor for therapeutic recombinant proteins and disease model animals. Previously, conventional methods without using artificial nuclease-dependent DNA cleavage system were used to produce such transgenic livestock, but their efficiency is known to be low. In the last decade, the development of artificial nucleases such as zinc-finger necleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas has led to more efficient production of knock-out and knock-in transgenic livestock. However, production of knock-in livestock is poor. In mouse, genetically modified mice are produced by coinjecting a pair of knock-in vector, which is a donor DNA, with a artificial nuclease in a pronuclear fertilized egg, but not in livestock. Gene targeting efficiency has been increased with the use of artificial nucleases, but the knock-in efficiency is still low in livestock. In many research now, somatic cell nuclear transfer (SCNT) methods used after selection of cell transfected with artificial nuclease for production of transgenic livestock. In particular, it is necessary to develop a system capable of producing transgenic livestock more efficiently by co-injection of artificial nuclease and knock-in vectors into fertilized eggs.

• Journal of Microbiology and Biotechnology
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• 제25권2호
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• pp.280-287
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• 2015

Current influenza vaccines are produced in embryonated chicken eggs. However, egg-based vaccines have various problems. To address these problems, recombinant protein vaccines have been developed as new vaccine candidates. Unfortunately, recombinant proteins frequently encounter aggregation and low stability during their biogenesis. It has been previously demonstrated that recombinantly expressed proteins can be greatly stabilized with high solubility by fusing stabilizing peptide (SP) derived from the C-terminal acidic tail of human synuclein (ATS). To investigate whether SP fusion proteins can induce protective immunity in mice, we produced influenza HA and SP fusion protein using a baculovirus expression system. In in vitro tests, SP-fused recombinant HA1 (SP-rHA1) was shown to be more stable than recombinant HA1 (rHA1). Mice were immunized intramuscularly with baculovirus-expressed rHA1 protein or SP-rHA1 protein ($2{\mu}g/mouse$) formulated with aluminum hydroxide. Antibody responses were determined by ELISA and hemagglutination inhibition assay. We observed that SP-rHA1 immunization elicited HA-specific antibody responses that were comparable to rHA1 immunization. These results indicate that fusion of SP to rHA1 does not negatively affect the immunogenicity of the vaccine candidate. Therefore, it is possible to apply SP fusion technology to develop stable recombinant protein vaccines with high solubility.