• 전기학회논문지
• /
• 제56권12호
• /
• pp.2208-2213
• /
• 2007

In this study we have developed a hyperspectrum imaging system for highly sensitive and effective imaging analysis. An optical setup was designed using acoustic optical tunable filter (AOTF) for high sensitive hyperspectrum imaging. Light emitted by mercury lamp gets split in to diffracted and undiffracted beams while passing though AOTF. GFP transfected HEK-293 cell line was used as a model for in vitro imaging analysis. Cells were first, analyzed by fluorescence microscope followed by flow cytometric analysis. Flow cytometric analysis showed 66.31% transfection yield in GFP transfected HEK-293 cells. Various images of GFP transfected HEK-293 cell were grabbed by collecting the diffracted light using a CCD over a dynamic range of frequency of 129-171 MHz with an interval of 3 MHz. Subsequently, for in vivo image analysis of GFP transfected cells in mouse, a whole-body-imaging system was constructed. The blue light of 488 nm wavelength was obtained from a Xenon arc lamp using an appropriate filter and transmitted through an optical cable to a ring illuminator. To check the efficacy of the newly developed whole-body-imaging system, a comparative imaging analysis was performed on a normal mouse in presence and absence of Xenon arc irradiation. The developed hyperspectrum imaging analysis with AOTF showed the highest intensity of green fluorescent protein at 153 MHz of frequency and 494 nm of wavelength. However, the fluorescence intensity remained same as that of the background below 138 MHz (475 nm) and above 162 MHz (532 nm). The mouse images captured using the constructed whole-body-imaging system appeared monochromatic in absence of Xenon arc irradiation and blue when irradiated with Xenon arc lamp. Nevertheless, in either case mouse images appeared clearly.

• Animal cells and systems
• /
• 제5권3호
• /
• pp.253-262
• /
• 2001

Ligand-receptor clustering event is the most important step in leukocyte adhesion and spreading on endothelial cells. Intercellular adhesion molecule-1 (ICAM-1) has been shown to enhance leukocyte adhesion, but the molecular event during the process of adhesion is unclear. To visualize the dynamics of ICAM-1 movement during adhesion, we have engineered stable Chinese hamster ovary cell lines expressing ICAM-1 fused to a green fluorescent protein (IC1_GFP/CHO) and examined them under the fluorescence microscopy. The transfection of IC1_GFP alone in these cells was sufficient to support the adhesion of K562 cells that express $\alpha$L$\beta$2 (LFA-1) integrin stimulated by CBR LFA-1/2 mAb. This phenomenon was mediated by ICAM-1-LFA-1 interactions, as an mAb that specifically inhibits ICAM-1-LFA-1 interaction (RRl/l) completely abolished the adhesion of LFA-1＊ cells to IC1_ GFP/CHO cells. We found that the characteristic of adhesion was followed almost immediately (~10 min) by the rapid accumulation of ICAM-1 on CHO cells at a tight interface between the two cells. Interestingly, ICI_GFP/CHO cells projected plasma membrane and encircled approximately half surface of LFA-1+ cells, as defined by confocal microscopy. This unusual phenomenon was also confirmed on HUVEC after adhesion of LFA-1＊ cells. Neither cytochalasin D nor 2,3-butanedione 2-monoxime an inhibitor of myosin light chain kinase blocked LFA-1-mediated ICAM-1 clustering, indicating that actin cytoskeleton and myosin-dependent contractility are not necessary for ICAM-1 clustering. Taken together, we suggest that leukocyte adhesion to endothelial cells induces specialized form of ICAM-1 clustering that is distinct from immunological synapse mediated by T cell interaction with antigen presenting cells.

• Reproductive and Developmental Biology
• /
• 제29권1호
• /
• pp.57-62
• /
• 2005

본 연구에서는 retrovirus를 이용한 유전자 전이에 있어서 대두되는 큰 문제점의 하나인 외래 유전자의 지속적인 발현으로 인한 개체의 생리적인 손상을 최소화하기 위하여 tetracycline계 물질의 공급 여부에 따라서 발현을 유도적으로 조절할 수 있는 one vector 형태의 Tet-On system을 구축하고자 하였다. 또한 WPRE 서열을 이 vector 상에 도입하여 유도적 조건에서 외래 유전자의 발현이 보다 강하게 일어날 수 있는 효율적인 retrovirus vector system을 확립하고자 하였다. 구축한 각각의 vector system에서 fluorometry와 western blotting을 이용하여 GFP 유전자의 발현 정도를 비교 측정한 결과, RevTRE-EGFP-WPRE-RSVp-rtTA2SM2 virus를 이용하여 유전자를 전이시킨 표적세포에서 GFP의 절대적인 발현량이 가장 큰 것으로 나타났고, 유전자 발현의 turn on/off에 의한 유도율은 RevTRE-EGFP-RSVp-rtTA2SM2-WPRE virus의 경우에서 8∼21배로 가장 높은 것으로 나타났다. 이상의 결과에서 외래 유전자의 발현을 효율적으로 조절할 수 있는 vector system은 WPRE가 rtTA2SM2 서열의 3에 위치한 형태로, 이 system을 이용하여 생산한 고감염가의 virus는 유전자 치료나 형질전환 동물의 생산에 있어서 요구되는 외래 유전자의 발현을 효율적으로 조절할 수 있는 수단이 될 것이다.

• Journal of Microbiology and Biotechnology
• /
• 제27권6호
• /
• pp.1106-1111
• /
• 2017

Escherichia coli was engineered to sense methanol by employing a chimeric two-component system (TCS) strategy. A chimeric MxaY/EnvZ (MxaYZ) TCS was constructed by fusing the Paracoccus denitrificans MxaY with the E. coli EnvZ. Real-time quantitative PCR analysis and GFP-based fluorescence analysis showed maximum transcription of ompC and the fluorescence at 0.01% of methanol, respectively. These results suggested that E. coli was successfully engineered to sense methanol by the introduction of chimeric MxaYZ. By using this strategy, various chimeric TCS-based bacterial biosensors can be constructed and used for the development of biochemical-producing recombinant microorganisms.

• 대한약학회:학술대회논문집
• /
• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
• /
• pp.145.3-146
• /
• 2003

Transgenic Eleutherococcus senticosus plants were prepared by introducing the genes for squalene synthase (SQS), hygromycin phosphotransferase (HPT) and green fluorescent Protein (GFP) through Agrobacterium-mediated transformation. The enzyme, SQS, represents a putative branch point in the isoprenoid pathway capable of diverting carbon flow specifically to the biosynthesis of phytosterol and oleanolic acid. The full SQS gene was isolated from P. ginseng roots. Early globular embryo clusters developed from embryogenic callus were used as the explant source. (omitted)

• Asian-Australasian Journal of Animal Sciences
• /
• 제28권12호
• /
• pp.1721-1728
• /
• 2015

Embryonic stem cells (ESCs) have been used as a powerful tool for research including gene manipulated animal models and the study of developmental gene regulation. Among the critical regulatory factors that maintain the pluripotency and self-renewal of undifferentiated ESCs, NANOG plays a very important role. Nevertheless, because pluripotency maintaining factors and specific markers for livestock ESCs have not yet been probed, few studies of the NANOG gene from domestic animals including bovine have been reported. Therefore, we chose mouse ESCs in order to understand and compare NANOG expression between bovine, human, and mouse during ESCs differentiation. We cloned a 600 bp (-420/+181) bovine NANOG 5'-flanking region, and tagged it with humanized recombinant green fluorescent protein (hrGFP) as a tracing reporter. Very high GFP expression for bovine NANOG promoter was observed in the mouse ESC line. GFP expression was monitored upon ESC differentiation and was gradually reduced along with differentiation toward neurons and adipocyte cells. Activity of bovine NANOG (-420/+181) promoter was compared with already known mouse and human NANOG promoters in mouse ESC and they were likely to show a similar pattern of regulation. In conclusion, bovine NANOG 5-flanking region functions in mouse ES cells and has characteristics similar to those of mouse and human. These results suggest that bovine gene function studied in mouse ES cells should be evaluated and extrapolated for application to characterization of bovine ES cells.

• 한국수정란이식학회지
• /
• 제17권2호
• /
• pp.109-115
• /
• 2002

The present study was conducted for the production of transgenic cloned cows those secrete human lactoferricin into milk by somatic cell nuclear transfer (NT). To estimate detrimental effects of gene transfection on transgenic cloned embryo production, development rates of NT embryos were compared between transfected and non-transfected cumulus and ear fibroblast cells. An expression plasmid for human lactofericin (pbeta-LFC) was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and human lactoferricin target gene into a pcDNA3 plasmid. Two bovine somatic cell lines (cumulus cell and ear fibroblast) were established and transfected with the expression plasmid using a liposomal transfection reagent, Fugene6 as a carrier. Cumulus cell and ear fibroblast were transfected at the passage of 2 to 4, trypsinized and GFP-expressing cells were randomly selected and used for somatic cell NT. Developmental competences (rates of fusion, cleavage, and blastocyst formation) in bovine transgenic somatic cell NT embryos reconstructed with non-transfectecd cells were significantly higher than those from transfected cells in cumulus cell and ear fibroblast (P＜0.05). This study indicated that transfection of done. cell has detrimental effect on embryo development in bovine transgenic NT.

• 한국수정란이식학회:학술대회논문집
• /
• 한국수정란이식학회 2002년도 국제심포지엄
• /
• pp.74-74
• /
• 2002

Production of u 1-antitrypsin ($\alpha$AT) in transgenic cows has a great value in the field of medicine. The present study was conducted to determine the effect of chemically defined KSOM media on in vitro development of bovine transgenic nuclear transfer (NT) embryos. An expression plasmid for human $\alpha$AT was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and a human $\alpha$AT target gene into a pcDNA3 plasmid. Cumulus cells as donor nuclei in NT were collected from a Holstein cow and transfected by lipid-mediated method using FuGene6 (Roche Molecular Biochemicals, USA) as reagent. GFP expressed cumulus cells were introduced into recipient oocytes under DIC microscopy equipped with FITC filter set. After electrical fusion and chemical activation, reconstructed embryos were cultured in 1) SOF + 0.8% BSA, 2) KSOM + 0.8% BSA, 3) KSOM + 10% FBS and 4) KSOM +0.01% PVA for 192 h at 39$^{\circ}C$ with 5% $CO_2$, 5% $O_2$ and 90% $N_2$in humidified condition. The development of the embryos was recorded and the GFP expression in blastocyst was determined under FITC filter. The average fusion rate was 73.8% (251/340; n=8). The development rates to 2-4 cells, morula, blastocysts and expression rates in blastocysts varied from 70.3 to 76.5%, 30.2 to 33.8%, 25.4 to 33.8% and 11.8 to 15.6%, respectively. The difference in development and expression rates of embryos among 4 culture groups was not significant (P>0.05). This study indicates that chemically defined KSOM medium is also able to support development of bovine transgenic NT embryos at similar rate of SOF or KSOM supplemented with BSA or serum.

• 한국정보통신학회:학술대회논문집
• /
• 한국정보통신학회 2019년도 춘계학술대회
• /
• pp.533-536
• /
• 2019

재조합 배큘로 바이러스는 배양 된 곤충 세포에서 이종 유전자를 발현하는데 널리 사용된다. 재조합 배큘로 바이러스는 광범위한 포유류 세포 유형에서 재조합 단백질의 발현을위한 유전자 전달 벡터로서 작용할 수있다. 바큘로 바이러스 시스템은 안전성, 대규모 및 높은 수준의 유전자 발현 관점에서 중요한 이점을 갖는다. 본 연구에서는 pOPINEneo-3C-GFP 벡터로부터 재구성 된 바큘로 바이러스 벡터를 사이토 메갈로 바이러스 (CMV) 프로모터, 강화 된 녹색 형광 단백질 (EGFP) 및 p53과 NcoI 및 XhoI로 재조합시켰다. 이러한 재조합 벡터를 다양한 세포 및 세포주에 감염시켰다. 이와 같이 개발 된 바큘로 바이러스 벡터는 재조합 유전자의 전이 및 발현을 통상적 인 벡터와 비교하여 분석 하였다. 이러한 결과는 바큘로 바이러스 벡터가 대조군 벡터보다 전이 및 전이에서 더 높은 효율을 갖는다는 것을 시사한다. 본 연구는 과학 기술부, 한국 정보 기술 진흥 기금 (MSIP)이 후원하는 한국 연구 재단(NRF)을 통해 중견 연구원 프로그램 (NRF-2016R1A2B4016552)을 통해 지원되었다.

• Asian-Australasian Journal of Animal Sciences
• /
• 제13권5호
• /
• pp.587-594
• /
• 2000

At present embryonic stem (ES) cells with confirmed pluripotential properties are only available in the mouse. Recently, we were able to isolate, culture and genetically transform primordial germ cell (PGC)-derived cells from pig embryos and demonstrate their ability to contribute to chimera development in the pig. In order to determine whether the system we developed could be used to isolate embryonic germ (EG) cells from other mammalian species, we placed isolated PGCs from cattle, goats, rabbits and rats in culture. Briefly, PGCs were isolated from fetuses of cow (day 30-50), goat (day 25), rabbit (day 15-18) and rat (day 11-12), and plated on STO feeder cells in Dulbecco's modified Eagle's medium (DMEM): Ham's F10 medium (1:1) supplemented with 0.01 mM nonessential amino acids, 2 mM L-glutamine, 0.1 mM $\beta$ - mercaptoethnol, soluble recombinant human stem cell factor (SCF; 40ng/ml), human basic fibroblast growth factor (bFGF; 20ng/ml) and human leukemia inhibitory factor (LIF; 20ng/ml). For maintenance of the cells, colonies were passed to fresh feeders every 7-10 days. In all species tested, we were able to obtain and maintain colonies with ES-like morphology. Their developmental potential was tested by alkaline phosphatase (AP) staining and in vitro differentiation assay. For genetic transformation, cells were electroporated with a construct containing the green fluorescent protein (GFP) under the control of the cytomegalovirus (CMV) promoter. GFP-expressing colonies were detected in cattle, rabbits and rats. These results suggest that PGC-derived cells from cattle, goats, rabbits and rats can be isolated, cultured, and genetically transformed, and provide the basis for analyzing their developmental potential and their possible use for the precise genetic modification of these species.