• 대한수의학회지
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• 제53권4호
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• pp.217-224
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• 2013

Transgenic plants have been tested as an alternative host for the production and delivery of experimental oral vaccines. Here, we developed transgenic potatoes that express the major antigenic sites A and D of the glycoprotein S from transmissible gastroenteritis coronavirus (TGEV-$S_{0.7}$) under three expression vector systems. The DNA integration and mRNA expression level of the TGEV-$S_{0.7}$ gene were confirmed in transgenic plants by PCR and northern blot analysis. Antigen protein expression in transgenic potato was determined by western blot analysis. Enzyme-linked immunosorbent assay results revealed that based on a dilution series of Escherichia coli-derived antigen, the transgenic line P-2 had TGEV-$S_{0.7}$ protein at levels that were 0.015% of total soluble proteins. We then examined the immunogenicity of potato-derived TGEV-$S_{0.7}$ antigen in mice. Compared with the wild-type potato treated group and synthetic antigen treated group, mice treated with the potato-derived antigen showed significantly higher levels of immunoglobulin (Ig) G and IgA responses.

• Reproductive and Developmental Biology
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• 제35권3호
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• pp.197-201
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• 2011

This study was conducted to investigate the effects of donor cell treatments on the production of transgenic cloned piglets. Ear fibroblast cell obtained from NIH MHC Inbred minipig was used as control. The GalT knock-out/CD45 knock-in (GalT/CD46) transgenic cell lines were established and used as donor cells. The reconstructed GalT/CD46 embryos were surgically transferred into oviduct of naturally cycling surrogate sows (Landrace ${\times}$ Yorkshire) on the second day of standing estrus. Unlike control (1.2 kV/cm, 75.4%), the fusion rate of the GalT/CDl6 donor cells was significantly higher in 1.5 kV/cm, (84.5%) than that of 1.25 kV/cm, (20.2%) (p<0.01). When the number of the transferred embryos were more than 129, the pregnancy and delivery rates were increased to 13/20 (65%) and 5/20 (25%) compared to less then 100 group [1/6 (16.7%) and 0/6 (0%)], respectively. To analyze the effect of donor cell culture condition on pregnancy and delivery rates, the GalT/CD46 donor cells were cultured with DMEM or serum reduced medium. In serum reduced medium group, the pregnancy and delivery rates were improved to 8/12 (66.7%) and 5/12 (41.7%) compared to DMEM group [3/7 (42.9%) and 0/7 (0%)], respectively. In conclusion, it can be postulated that an appropriate fusion condition and culture system is essential factors to increase the efficiency of the production of transgenic cloned piglets.

• 생명과학회지
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• 제28권4호
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• pp.496-507
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• 2018

톡소이드는 독성은 제거되고 항원성은 유지시킨 독소 단백질로써, 다양한 병원체의 감염 및 질병 예방을 위해 지속적으로 연구 되었다. 그러나, 톡소이드의 활성 감소 및 이와 함께 사용하는 어쥬번트의 부작용 등이 지속적으로 보고되면서, 면역성은 강화하고 어쥬번트의 사용은 줄일 수 있는 톡소이드 항원 전달 시스템이 필요하게 되었다. 따라서, 이러한 단점을 개선하고자 최근 새로운 백신과 약물 전달수송을 위해 다양한 분야에서 활용하고 있는 마이크로/나노 운반체를 톡소이드 항원에 도입하고 있다. 이와 같은 마이크로/나노 운반체는 미생물 자체를 이용하거나 미생물을 통해 생산해 낼 수도 있으며, 더 나아가 다양한 소재의 폴리머를 이용하여 제작할 수 있다. 본 총설에서는 톡소이드 항원 전달을 위한 마이크로/나노 운반체를 미생물 유래의 ghost cells (GCs), 그람 음성 세균이 분비하는 outer membrane vesicles (OMVs) 및 고분자 폴리머로 구성된 nanoparticles (NPs)으로 분류하였다. 마지막으로 각 운반체에 대한 톡소이드 항원의 전달 방식 및 이를 적용하였을 때 일어나는 면역반응에 대하여 서술하였으며, 이를 통해 향후 톡소이드의 효율 및 부작용이 개선되기를 기대한다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제7권5호
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• pp.303-310
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• 2002

Cell therapy applied to wound healing or tissue regeneration presents a revolutionary realm to which principles of gene engineering and delivery may be applied. One promising application is the transplantation of cells into the wounded tissue to help the tissue repair. However, when cells are transplanted from in vitro to in vivo, immune rejection occurs due to the immune response triggered by the activation of T-cell, and the transplanted cells are destroyed by the attack of activated T-cell and lose their function. Immune suppressant such as FK506 is commonly used to suppress immune rejection during transplantation. However, such kind of immune suppressants not only suppresses immune rejection in the periphery of transplanted cells but also suppresses whole immune response system against pathogenic infection. In order to solve this problem, we developed a method to protect the desired cells from immune rejection without impairing whole immune system during cell transplantation. Previously, we reported the success of constructing glomerular epithelial cells for removal of immune complex, in which complement receptor of type 1 (CR1) was over-expressed on the membrane of renal glomerular epithelial cells and could bind immune complex of DNA/anti-DNA-antibody to remove immune complex through phagocy-tosis [1]. Attempting to apply the CR1-expressing cells to cell therapy and evade immune rejection during cell transplantation, we constructed three plasmids containing genes encoding a soluble fusion protein of cytolytic T lymphocyte associated antigen-4 (CTLA4Ig) and an enhanced green fluorescent protein (EGFP). The plasmids were transfected to the above-mentioned glomerular epithelial cells to express both genes simultaneously. Using the clone cells for cell transplantation showed that mice with autoimmune disease prolonged their life significantly as compared with the control mice, and two injections of the cells at the beginning of two weeks resulted in remarkable survivability, whereas it requires half a year and 50 administrations of proteins purified from the same amount of cells to achieve the same effect.

• 생명과학회지
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• 제22권6호
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• pp.703-712
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• 2012

AAV는 매우 안전하고 효율적인 유전자전달방법으로 인정되어 왔다. 그러나, AAV가 가진 생물의약품으로서 단점은 효율적이고 재현성 높은 생산법이 취약하고, 또한 다양한 혈청형을 간단한 한 가지 공통적 방법으로 신뢰성 있게 정량하는 방법이 개발되어야 하는 것이다. 따라서, 본 연구에서는 AAV2와 아데노바이러스를 동시에 감염하는 종래의 생산법에 의한 효율성과 새로운 생산법, 즉 AAV2 감염 후 pHelper 플라스미드를 transfection 하는 방법을 통한 생산효율성을 비교하였고, HEK293과 293T를 생산세포주로 하여 시간에 따른 생산효율성도 분석하였다. 그 결과 AAV2와 pHelper DNA를 포함한 새로운 생산법은 기존의 방법에 비해 10배 이상 높은 생산효율성을 보였고, 293T에서 AAV2를 10 MOI로 감염한 후 5일째에 가장 높은 생산효율성을 보였는데, 생산세포 한 개 당 $1.61{\times}10^5$ virus genomes (v.g.)을 생산하는 결과였다. 따라서 이 생산조건을 다른 혈청형 생산에 적용한 결과, 모든 혈청형에서 생산세포주 한 개 당 $10^5$ v.g. 이상을 생산하는 효율성을 보였다. 한편, 다양한 AAV 혈청형을 한 가지 공통적인 방법으로 정량하기 위해 the universal PCR 프라이머를 제작하였고, 그것을 이용하여 신뢰성 높고 10개 분자까지도 증폭이 가능한 결과를 모든 혈청형에서 얻었다. 그러므로 이 한 쌍의 정량용 the universal 프라이머는 임상시험용 아데노바이러스벡터에 존재하는 AAV오염을 검출하는 것에도 사용 가능하다.

• 대한핵의학회지
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• 제38권3호
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• pp.253-258
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• 2004

목적: 약물이나 유전자 전달에 이용되는 생체적합성이 높은 키토산의 간세포 지향성을 위해서 갈락토스를 수식하는 방법이 널리 이용되어지고 있다. 이번 연구에서는 갈락토스 수식 키토산의 간세포지향성 획득을 평가하는데 있어서 핵의학 영상법의 유용성에 대해 알아보고자 하였다. 대상 및 방법: 키토산에 NHS와 EDC를 이용하여 30 mol%의 lactobionic acid를 결합시켜 갈락토스 수식 키토산을 제조하였다. 얻어진 갈락토스 수식 키토산을 투석 시킨 후 동결 건조하여 얻어 낸 다음 키토산의 기본구조인 glucoseamine에 methyl기를 첨가시키는 반응을 하여 최종화합물을 합성하였다. GMC의 세포내 독성은 MTT assay를 통하여 확인하였다. 제조된 GMC에 $SnCl_2{\cdot}2H_2O$를 이용하여 $^{99m}Tc$을 표지하였다. 표지 후 안정성은 아세톤과 생리식염수를 이용하여 1시간까지 확인하였다. $^{99m}Tc$-GMC와 $^{99m}Tc$-MC 55.5 MBq (1.5 mCi)를 토끼의 외이정맥으로 주사 후, 감마카메라를 이용하여 10분, 30분, 60분, 90분 간격으로 전면 영상을 얻었다. 결과: 키토산에 lactobionic acid 30 mol%를 반응시켜 7.4 mol%의 galactose group이 키토산에 결합한 것을 확인하였다. 갈락토스 수식 키토산을 메틸화한 결과는 tri, di, mono가 각각 8.8%, 46%, 35.2%인 것을 확인하였다. MTT assay결과를 통해 GMC의 세포에 미치는 독성은 거의 없는 것을 확인할 수 있었다. 표지 효율은 메틸화시키지 않은 $^{99m}Tc$-GC가 아세톤과 생리식염수에서 각각 88%, 72%를 보인 반면에 $^{99m}Tc$-GMC는 96%정도를 보여 보다 높은 표지효율을 가지는 것을 확인하였다. $^{99m}Tc$-MC를 주사후 얻은 토끼 영상에서 키토산은 일반적으로 대부분 신장을 통해 배출되며, 간과 비장, 골격계에는 매우 적은 분포를 보이는데 비해. 갈락토스가 수식된 $^{99m}Tc$-GMC에서는 분포에 변화가 생겨 간, 신장, 그리고 방광에서 높은 방사능이 관찰되는 소견을 보였다. 결론: 핵의학 영상법은 갈락토스 리간드 수식 키토산의 간세포 지향성 여부를 평가하기 위한 생체내 평가법으로 이용될 수 있을 것으로 사료되었다.

• 한국고분자학회:학술대회논문집
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• 한국고분자학회 2006년도 IUPAC International Symposium on Advanced Polymers for Emerging Technologies
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• pp.263-263
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• 2006

Poly (ethylene glycol)(PEG) - Poly (${\varepsilon}-caprolactone(CL)$) - Poly (D,L lactide(LA) (PCLA-PEG-PCLA) was synthesized by ring-opening polymerization to form temperature sensitive hydrogel triblock copolymer. The triblock copolymer was acrylated by acryloyl chloride. ${\beta}-amino$ ester was used as a pH sensitive moiety, in this study ${\beta}$- amino ester obtained from 1,4-butandiol diacrylate, and 4, 4' trimethylene dipiperidine, it have pKb around 6.6. pH/temperature sensitive penta-block copolymer (PAE-PCL-PEG-PCL-PAE) was synthesized by addition polymerization from acrylated triblock copolymer, 1,4-butandiol diacrylate, and 4, 4' trimethylene dipiperidine. Their physicochemical properties of triblock and penta-block copolymers were characterized by $^1H-NMR$ spectroscopy and gel permeation spectroscopy. Sol-gel phase transition behavior of PAE-PCL-PEG-PCL-PAE block copolymers were investigated by remains stable method. Aqueous media of the penta-block copolymer (at 20 wt%) changed from a sol phase at pH 6.4 and $10^{\circ}C$ to a gel phase at pH 7.4 and $37^{\circ}C$. The sol-gel transition properties of these block copolymers are influenced by the hydrophobic/hydrophilic balance of the copolymers, block length, hydrophobicity, stereo-regularity of the hydrophobic of the block copolymer, and the ionization of the pH function groups in the copolymer depended on the changing of environmental pH, respectively. The degradation and the stabilization at pH 7.4 and $37^{\circ}C$, and the stabilization at pH 6.4 and $10^{\circ}C,\;5^{\circ}C,\;0^{\circ}C$, of the gel were determined. The results of toxicity experiment show that the penta block copolymer can be used for injection drug delivery system. The sol?gel transition of this block copolymer also study by in vitro test ($200{\mu}l$ aqueous solution at 20wt% polymer was injected to mouse). Insulin loading and releasing by in vitro test was investigated, the results showed that insulin can loading easily into polymer matrix and release time is around 14-16days. The PAE-PCL-PEG-PCL-PAE can be used as biomaterial for drug, protein, gene loading and delivery.

• Journal of Plant Biotechnology
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• 제30권2호
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• pp.115-121
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• 2003

In this study, plant regeneration and Agrobacterium tumefaciens-mediated transformation Kentucky bluegrass(Poa pratensis L.) were evaluated. Three different types of calli were produced depending on the combinations of growth regulators. They were non-friable brown or gray-colored callus (type I), compact, friable and yellow or white-colored callus (typeII), and soft, watery translucent callus with differentiated structure (typeIII). The highest regenerable organogenic callus (typeII) was obtained on the medium containing 1mg/L, 2,4-D and 0.1mg/L BA. Additionally, the production of typeII calli increased significantly when AgNO$_3$ was added to the callus induction and growth medium. The highest frequency of multiple shout formation from typeII callus was obtained on MS medium containing 1mg/L BA and 1mg/L Thidiazuron(TDZ). The organogenic calli(typeII) were inoculated with Agrobacterium tumefaciens strain EHA101 harboring the binary vector pIG121Hm with $\beta$-glucuronidase gene, and various factors were found to influence the transfer-DNA delivery efficiency. The highest transient GUS activity was observed on typeIIcallus. In the present work, we reported the first transient GUS activity of Kentucky bluegrass mediated by Agrobacterium tumefaciens. Our system may contribute to genetic improvement for breed-recalcirtrant grass species, Kentucky bluegrass.

• Molecular & Cellular Toxicology
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• 제3권3호
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• pp.195-197
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• 2007

Currently praziquantel is used for treatment of not only clonorchiasis but also other trematodes and cestodes. But cure rate of praziquantel is just 60-80% for most trematodes. It needs for the treatment-failed patients to have more drugs. The cause of failure of treatment is not studied. We just know that the blood level of praziquantel is severely different among the people. We guess that this factor may influence the results of treatment. In an endemic area of human clonorchiasis in Heilongjiang Providence, China, 78 subjects were selected for the study. Three doses of 25 mg/kg (total 75 mg/kg) of praziquantel were administered to 78 clonorchiasis patients. After three weeks of treatment, stool examination was undertaken once again to confirm the cured and uncured subjects. To analyze SNP (single nucleotide polymorphism) of CYP3A5 PS2-1, CYP3A5 PS2-2, and CYP3A5*6, PCR method was done with specifically designed primers. The mutation rates of all sites were not significant statistically. The number of subjects was too small, so we need more subjects and other delivery proteins of bile ducts (ex. MRP etc.) were also considered for effects of praziquantel. We analyzed, for the first time, the entire CYP3A5 gene in a French population, using a polymerase chain reaction- single strand conformational polymorphism (PCR-SSCP) strategy.

• Plant Biotechnology Reports
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• 제3권4호
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• pp.333-340
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• 2009

Developmental anomalies in the plumule meristem of peanut (Arachis hypogaea L.) somatic embryos resulted in poor shoot differentiation and reduced plant recovery. Existing meristems with caulogenic potential have never been tested for embryogenesis in peanut. The present experiment was designed to test the mature zygotic embryo axis derived plumule with three meristems for somatic embryogenesis. Embryogenic masses and embryos developed from the caulogenic meristems in the axils. Exposure of 2 weeks in primary medium with $90.5{\mu}M$ 2,4-D suppressed the shoot tip differentiation temporarily which then regained the ability to form the shoot on withdrawal of 2,4-D. Exposure of 4 weeks in primary medium with $90.5{\mu}M$ 2,4-D suppressed the shoot tip differentiation irreversibly. No shoot formation was noted from the tips in any of the cultures which were in secondary medium with $13.6{\mu}M$ 2,4-D. Development of somatic embryos directly from axillary meristems was confirmed histologically. Conversion frequency of these embryos was 11%. Thus, in this report, we describe a method to obtain somatic embryos from the determined organogenic buds of the axillary meristem, by culturing the nodal explant vertically on embryo induction medium. It also displays the possibility of obtaining both embryogenic and organogenic potential in two parts of the same explant simultaneously. The possibility of extending this approach for genetic transformation in in vivo system through direct DNA delivery or Agrobacterium injection in meristems can also be explored. Using Agrobacterium rhizogenes, we have demonstrated the possibility of gene transfer in the axillary meristems of seed-derived plumule explant.