• 한국수정란이식학회지
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• 제19권3호
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• pp.245-255
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• 2004

포유동물 난자의 체외수정은 외래유전자 도입에 의한 형질전환동물 생산과 우수한 형질을 가진 개체의 보존, 인간의 불임연구 등과 같은 수정란이식 기술로서 널리 이용되고 있다. 돼지 난포란을 이용한 체외수정란의 생산은 초기단계인 체외성숙 기술의 미확립, 그로인한 체외수정 시 높은 다정자 침입율과 불완전한 웅성전핵 형성 몇 체외발달능 정지현상(cell blocking) 등 어려움 때문에 아직도 다른 가축보다 양질의 수정란을 생산하기가 어려운 것으로 알려져 있다. 이와 같은 것을 해결하기 위하여 많은 연구자들은 배양액내에 hormon, growth factor, antioxdants 등과 같은 외인성 인자들을 첨가하고 있다. 이들 인자 중 antioxdant는 free radical을 소거하고 과산화물 생성을 억제하여 난자를 산화적 스트레스로부터 보호한다. 따라서 본 연구는 돼지 난포란으로부터 체외수정란의 생산에 있어서 배양액내 cysteine, catalase 및 glutathione의 첨가가 체외성숙, 체외수정 및 체외배양에 어떤 영향을 미치는지를 검토하였다. 실험 1은 체외성숙용 배양액인 TCM-199 용액에 catalase(100, 200, 500U/$m\ell$)와 glutathione(0.5, 1.0, 1.5mM/$m\ell$)의 첨가, 실험 2는 성숙된 난자의 체외수정용 배양액인 mTBM 용액에 cysteine(0.1, 1.6, 1.0mM/$m\ell$), catalase(100, 200, 500U/$m\ell$)와 glutathione(0.5, 1.0, 1.5mM/$m\ell$)의 첨가, 실험 3은 체외성숙 및 체외수정된 난자의 체외배양용 배양액인 NCSU-23 용액에 cysteine(0.1, 1.6, 1.0mM/$m\ell$), catalase(100, 200, 500U/$m\ell$)와 glutathione(0.5, 1.0, 1.5mM/$m\ell$)을 첨가하여 배반포로의 배 발달율을 관찰하였던 결과는 다음과 같다. 1. 체외성숙시 catalase의 경우는 500U 첨가군의 27.2%로 무첨가군의 15.4%보다 유의하게 높았다(p<0.05). 한편 glutathione의 경우 배반 포로의 배 발달율은 무첨가군과의 차이는 없었다. 그러나 1.0mM 첨가군에서 상실배까지의 배 발달율인 72%는 무첨가군의 53.9%보다 유의하게 높았다(p<0.05). 2. 체외수정시 여러 종류의 항산화제 첨가는 첨가하는 농도와 관계없이 배반포로의 배 발달율은 무첨가군과 차이가 없었다. 3. 체외배양시 여러 종류의 항산화제 첨가는 첨가하는 농도와 관계없이 배반포로의 배 발달율은 무첨가군과 차이가 없었다. 이상의 결과를 종합적으로 하면 돼지 난포란을 이용한 배반포의 체외생산에 있어서 배양액내 항산화제의 첨가는 체외성숙단계에서만 효과적이었다. 이것은 아마도 항산화제가 체외성숙 시 난포란 내에서 일어나는 여러 가지 생화학 반응의 처리시간과 관련하여 활성화시킴으로써 난포란의 생존력을 높인 것이라고 사료되기 때문에 앞으로는 돼지 난포란의 효율적인 체외성숙에 대해서 배양액내 첨가물질은 물론 나아가서 방법론적인 측면에서 더욱 연구되어져야 할 것이다.

• Journal of Plant Biotechnology
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• 제1권1호
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• pp.31-38
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• 1999

An artificial symbiosis was established between diazotropic Azomonas insignis and strawberry (Fragaria x ananassa). The partnership was created by in vitro techniques through callus induction and organogenesis. The basis of this partnerships is the bacterial dependence on the plants metabolic activity, using maltose in the medium as a carbon and energy source which can be utilized by the plant cells only. The presence of bacteria in the intercellular spaces of the callus tissues and regenerated plants was proven by microscopic techniques. Nitrogenase activity could also be detected in the plant tissues. For successful and high frequency introduction of bacteria to the plant tissues, biolistic gun method was used. On the basis of the DNA transfer method, Azotobacter vinelandii bacteria were delivered directly into strawberry tissues by the particle bombardment. This was the first use of living bacteria as microprojectils for bombardment of plant tissues. The treatment was successful, the presence of bacteria in the developing callus tissue and regenerated plants were detected by light and electron microscopy.

• 한국환경보건학회지
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• 제44권1호
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• pp.31-43
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• 2018

Objectives: Effective genetic toxicology and molecular biology research techniques and strategies that are highly correlated with the carcinogenic inhalation toxicity test and related research are required. The aim of this study was to maximize the utilization of chemical substances to prevent workers' occupational diseases. Methods: We surveyed the literature, domestic and international references, and the status of relevant domestic and foreign professional organizations. Expert advisory opinions were reflected, and experts were consulted by participating in domestic and overseas academic conferences. Results: The current status of domestic and international genotoxic toxicity evaluation was examined through various documents from related organizations. Cell models for in vitro lung toxicology were investigated and summarized, and the human resources and performance results of genetic toxicity studies and pilot projects were compared and analyzed by holding an advisory meeting. We examined domestic and international genotoxicity guidelines and investigated new test methods for the development of genotoxicity and carcinogenicity. Ultimately, we described long-term future predictions, including the implementation of our researchers' recommendations and occupational genetic toxicology forecasts for future worker health protection. Conclusions: This research project aims to establish current genetic toxicology and molecular biology research techniques and strategies that can maximize the linkage with the carcinogenic inhalation toxicity test and research in the future. We expanded the study of genetic toxicity and establish a foundation forgenetic toxicity in accordance with research trends in Korea and abroad.

• Journal of Animal Science and Technology
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• 제63권2호
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• pp.281-294
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• 2021

Although somatic cell nuclear transfer (SCNT) is frequently employed to produce cloned animals in laboratories, this technique is expensive and inefficient. Therefore, the handmade cloning (HMC) technique has been suggested to simplify and advance the cloning process, however, HMC wastes many oocytes and leads to mitochondrial heteroplasmy. To solve these problems, we propose a modified handmade cloning (mHMC) technique that uses simple laboratory equipment, i.e., a Pasteur pipette and an alcohol lamp, applying it to porcine embryo cloning. To validate the application of mHMC to pig cloning, embryos produced through SCNT and mHMC are compared using multiple methods, such as enucleation efficiency, oxidative stress, embryo developmental competence, and gene expression. The results show no significant differences between techniques except in the enucleation efficiency. The 8-cell and 16-cell embryo developmental competence and Oct4 expression levels exhibit significant differences. However, the blastocyst rate is not significantly different between mHMC and SCNT. This study verifies that cloned embryos derived from the two techniques exhibit similar generation and developmental competence. Thus, we suggest that mHMC could replace SCNT for simpler and cheaper porcine cloning.

• Mass Spectrometry Letters
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• 제11권2호
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• pp.36-40
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• 2020

Untargeted metabolomics is a useful tool for drug development focusing on novel chemotherapeutic and chemopreventative agents against cancer cells. In recent years, quadrupole time of flight liquid chromatography-mass spectrometry (Q-TOF LC/MS)-based untargeted metabolomic approaches have gained importance to evaluate the effect of these agents at the molecular level. The researchers working on cell culture studies still do not apply standardized methodologies on sample preparation for untargeted metabolomics approaches. In this study, the rough and wet lysis techniques performed on MCF-7 breast cancer cells were compared with each other via the Q-TOF LC/MS-based metabolomic approach. The C18 and hydrophilic interaction liquid chromatography (HILIC) columns were used for the separation of the metabolites in MCF-7 cell lysates. 505 peaks were detected through the HILIC column and 551 peaks were found through the C18 column for the wet lysis technique. This situation supported by the base peak chromatograms showed that the wet lysis technique allowed us to extract higher number of non-polar metabolites. Almost equal number of metabolites was found for the C18 and HILIC columns (697 peaks for the HILIC column and 695 peaks for the C18 column) when the rough lysis technique was used. However, the intensities of polar metabolites were higher for the rough lysis technique on base peak chromatograms for both the HILIC and C18 columns. Although cell lysis technique, which is the first step in the sample preparation for cell culture studies, does not cause dramatic differences in the number of the detected metabolite peaks, it affects the polar and non-polar metabolite ratio significantly. Therefore, it must be considered carefully especially for in vitro drug development studies.

• Journal of Pharmaceutical Investigation
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• 제30권3호
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• pp.191-199
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• 2000

Genomics is providing targets faster than we can validate them and combinatorial chemistry is providing new chemical entities faster than we can screen them. Historically, the drug discovery cascade has been established as a sequential process initiated with a potency screening against a selected biological target. In this sequential process, pharmacokinetics was often regarded as a low-throughput activity. Typically, limited pharmacokinetics studies would be conducted prior to acceptance of a compound for safety evaluation and, as a result, compounds often failed to reach a clinical testing due to unfavorable pharmacokinetic characteristics. A new paradigm in drug discovery has emerged in which the entire sample collection is rapidly screened using robotized high-throughput assays at the outset of the program. Higher-throughput pharmacokinetics (HTPK) is being achieved through introduction of new techniques, including automation for sample preparation and new experimental approaches. A number of in vitro and in vivo methods are being developed for the HTPK. In vitro studies, in which many cell lines are used to screen absorption and metabolism, are generally faster than in vivo screening, and, in this sense, in vitro screening is often considered as a real HTPK. Despite the elegance of the in vitro models, however, in vivo screenings are always essential for the final confirmation. Among these in vivo methods, cassette dosing technique, is believed the methods that is applicable in the screening of pharmacokinetics of many compounds at a time. The widespread use of liquid chromatography (LC) interfaced to mass spectrometry (MS) or tandem mass spectrometry (MS/MS) allowed the feasibility of the cassette dosing technique. Another approach to increase the throughput of in vivo screening of pharmacokinetics is to reduce the number of sample analysis. Two common approaches are used for this purpose. First, samples from identical study designs but that contain different drug candidate can be pooled to produce single set of samples, thus, reducing sample to be analyzed. Second, for a single test compound, serial plasma samples can be pooled to produce a single composite sample for analysis. In this review, we validated the issue whether the second method can be applied to practical screening of in vivo pharmacokinetics using data from seven of our previous bioequivalence studies. For a given drug, equally spaced serial plasma samples were pooled to achieve a 'Pooled Concentration' for the drug. An area under the plasma drug concentration-time curve (AUC) was then calculated theoretically using the pooled concentration and the predicted AUC value was statistically compared with the traditionally calculated AUC value. The comparison revealed that the sample pooling method generated reasonably accurate AUC values when compared with those obtained by the traditional approach. It is especially noteworthy that the accuracy was obtained by the analysis of only one sample instead of analyses of a number of samples that necessitates a significant man-power and time. Thus, we propose the sample pooling method as an alternative to in vivo pharmacokinetic approach in the selection potential lead(s) from combinatorial libraries.

• Asian-Australasian Journal of Animal Sciences
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• 제29권2호
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• pp.230-240
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• 2016

Information on the effects of different yeast species on ruminal fermentation is limited. This experiment was conducted in a $3{\times}4$ factorial arrangement to explore and compare the effects of addition of three different live yeast species (Candida utilis 1314, Saccharomyces cerevisiae 1355, and Candida tropicalis 1254) at four doses (0, $0.25{\times}10^7$, $0.50{\times}10^7$, and $0.75{\times}10^7$ colony-forming unit [cfu]) on in vitro gas production kinetics, fiber degradation, methane production and ruminal fermentation characteristics of maize stover, and rice straw by mixed rumen microorganisms in dairy cows. The maximum gas production (Vf), dry matter disappearance (IVDMD), neutral detergent fiber disappearance (IVNDFD), and methane production in C. utilis group were less (p<0.01) than other two live yeast supplemented groups. The inclusion of S. cerevisiae reduced (p<0.01) the concentrations of ammonia nitrogen ($NH_3$-N), isobutyrate, and isovalerate compared to the other two yeast groups. C. tropicalis addition generally enhanced (p<0.05) IVDMD and IVNDFD. The $NH_3$-N concentration and $CH_4$ production were increased (p<0.05) by the addition of S. cerevisiae and C. tropicalis compared with the control. Supplementation of three yeast species decreased (p<0.05) or numerically decreased the ratio of acetate to propionate. The current results indicate that C. tropicalis is more preferred as yeast culture supplements, and its optimal dose should be $0.25{\times}10^7$ cfu/500 mg substrates in vitro.

• Asian-Australasian Journal of Animal Sciences
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• 제24권6호
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• pp.781-790
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• 2011

The effects of seedtime and maturity stage on nutritive value of five maize stover varieties, including conventional maize (Kexiangyu 11, CM), fodder maize (Huqing 1, FM), high oil maize (Gaoyou 115, HOM), sweet maize (Kexiangtianyu 1, SM) and waxy maize (Kexiangluoyu 1, WM), were examined based on chemical composition, in vitro gas production and in situ incubation techniques. Maize stover was sampled at d 17 and d 30 after tasseling, and designated as maturity stage 1 and stage 2, respectively. The average dry matter (DM) organic matter (OM), crude protein (CP) and fiber contents were the greatest for HOM, SM and FM, respectively. CM had the highest in vitro organic matter disappearance (IVOMD) and volatile fatty acid (VFA) concentration. The highest ammonia nitrogen ($NH_3$-N) concentration in the incubation solution, and effective degradability of DM ($ED_{DM}$) and neutral detergent fiber ($ED_{NDF}$) were observed in SM. Advanced maturity stage increased (p<0.05) DM content, $ED_{DM}$ and $ED_{NDF}$, but decreased (p<0.05) OM and CP contents, and decreased (p<0.05) b and a+b values, IVOMD and molar proportion of valerate in the incubation solution for maize stover. Maize sown in summer had greater (p<0.05) OM content, but lower DM, CP, neutral detergent fiber (NDF) and acid detergent fiber (ADF) content compared with maize sown in spring. Maize sown in summer had greater (p<0.001) IVOMD, $NH_3$-N concentration in the incubation solution and $ED_{NDF}$, but lower (p<0.01) ratio of acetate to propionate compared to maize sown in spring. The interaction effect of variety${\times}$seedtime was observed running through almost all chemical composition, in vitro gas production parameters and in situ DM and NDF degradability. The overall results suggested that SM had the highest nutrient quality, and also indicated the possibility of selecting maize variety and seedtime for the utilization of maize stover in ruminants.

• 대한약학회:학술대회논문집
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• 대한약학회 2002년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2
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• pp.201-205
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• 2002

It is generally accepted that the immune system is one of the major target organs for many toxic chemicals. In addition, many toxic chemicals require metabolic activation by cytochrome P450s for their toxicity. Although the immune cells possess a limited amount of drug metabolizing capacity, metabolic activation of certain toxicants in liver and immune organs may have a significant role in immunosuppression. In the present studies, the possible role of metabolic activation by cytochrome P450s in chemical-induced immunosuppression was reviewed, with a particular emphasis on the methodological techniques to detect immunotoxicants requiring metabolic activation in vivo and in vitro. (omitted)

• 대한핵의학회지
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• 제38권2호
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• pp.131-139
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• 2004

Small animal models are extensively utilized in the study of biomedical sciences. Current animal experiments and analysis are largely restricted to in vitro measurements and need to sacrifice animals to perform tissue or molecular analysis. This prevents researchers from observing in vivo the natural evolution of the process under study. Imaging techniques can provide repeatedly in vivo anatomic and molecular information noninvasively. Small animal imaging systems have been developed to assess biological process in experimental animals and increasingly employed in the field of molecular imaging studies. This review outlines the current developments in nuclear medicine imaging instrumentations including fused multi-modality imaging systems for small animal imaging.