• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.232-234
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• 2002

Bacillus cereus secretes a nonspecific phospholipase C (PLC) that catalyzes the hydrolysis of phospholipids to yield diacylglycerol and a phosphate monoester. This study focuses on the production of PLC by B. cereus and recombinant E. coli with fusion protein gene (plc::gfp). Fermentation processes have been monitored by a 2-dimensional fluorescence sensor.

• Korean Journal of Animal Reproduction
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• v.26 no.4
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• pp.347-351
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• 2002

Transgenes in HSY-TK gene driven by the lck promoter was tested for the expression in immune cells (Jurkat cells) to apply xenotransplantation of human cells into transgenic animals for the potential use of the proliferation or differentiation of human stem cells in the large animal such as an pig. Also, lck-CFP gene was used for transfection experiment into Jurkat cell to confirm the proper regulation of lck promoter for transgene expression in the T cells. Transfection of lck-GFP gene into Jurkat ceils induced CFP expression in transfected cells. The expression of Ick-TK and Ick-CFP genes was confirmed by RT-PCR using RNAs extracted from Jurkat cells, When Jurkat cells transfected with TK and CFP genes were selected against G418 or gancyclovir treatments, Jurkat cells transfected with TK gene were not proliferated in G4i8 and gancyclovir medium while intact cells or cells transfected with CFP gene could grow in gancyclovir medium. However, Jurkat cells transfected with TK or GFP gene were proliferated in G418 medium probably due to Neo$^{r}$ gene in the vector. Gancyclovir treatment destroyed Jurkat cells expressing TK gene indicating that T-cells expressing TK gene can be selectively eliminated by TK gene expression driven by lck promoter.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.6
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• pp.886-892
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• 2017

Objective: Transgenic technology is widely used for industrial applications and basic research. Systems that allow for genetic modification play a crucial role in biotechnology for a number of purposes, including the functional analysis of specific genes and the production of exogenous proteins. In this study, we examined and verified the cumate-inducible transgene expression system in chicken DF1 and quail QM7 cells, as well as loxP element-mediated transgene recombination using Cre recombinase in DF1 cells. Methods: After stable transfer of the transgene with piggyBac transposon and transposase, transgene expression was induced by an appropriate concentration of cumate. Additionally, we showed that the transgene can be replaced with additional transgenes by co-transfection with the Cre recombinase expression vector. Results: In the cumate-GFP DF1 and QM7 cells, green fluorescent protein (GFP) expression was repressed in the off state in the absence of cumate, and the GFP transgene expression was successfully induced in the presence of cumate. In the cumate-MyoD DF1 cells, MyoD transgene expression was induced by cumate, and the genes controlled by MyoD were upregulated according to the number of days in culture. Additionally, for the translocation experiments, a stable enhanced green fluorescent protein (eGFP)-expressing DF1 cell line transfected with the loxP66-eGFP-loxP71 vector was established, and DsRed-positive and eGFP-negative cells were observed after 14 days of co-transfection with the DsRed transgene and Cre recombinase indicating that the eGFP transgene was excised, and the DsRed transgene was replaced by Cre recombination. Conclusion: Transgene induction or replacement cassette systems in avian cells can be applied in functional genomics studies of specific genes and adapted further for efficient generation of transgenic poultry to modulate target gene expression.

• Korean Journal of Microbiology
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• v.44 no.2
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• pp.105-109
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• 2008

We studied on the function and localization of fission yeast Schizosaccharomyces pombe Nup97p, which is homologous to nucleoporin Nic96p in budding yeast Saccharomyces cerevisiae. There was no effect on growth and $poly(A)^{+}$ RNA distribution of cells when nup97 gene was overexpressed. However, the haploid ${\Delta}nup97::kan^{r}$ null mutants confirmed extensive $poly(A)^{+}$ RNA accumulation in the nucleus, abnormal DNA distribution, and cessation of growth when nup97 expression was repressed. We determined the subcellular localization of Nup97 tagged at the N terminus or the C terminus with GFP. Both fusions complemented growth defect of ${\Delta}nup97::kan^{r}$ null mutants. An integrated version of the nup97-GFP fusion was constructed at the nup97 locus. Nup97-GFP fusions expressed from its own promoter was localized at the nuclear periphery with a punctate appearance. These results suggest that Nup97p in fission yeast is also nucleoporin, which is involved in mRNA export.

• Animal Bioscience
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• v.36 no.6
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• pp.973-979
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• 2023

Objective: The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, which is the most efficient and reliable tool for precisely targeted modification of the genome of living cells, has generated considerable excitement for industrial applications as well as scientific research. In this study, we developed a gene-editing and detection system for chick embryo sexing during the embryonic stage. Methods: By combining the CRISPR/Cas9 technical platform and germ cell-mediated germline transmission, we not only generated Z chromosome-targeted knockin chickens but also developed a detection system for fluorescence-positive male chicks in the embryonic stage. Results: We targeted a green fluorescent protein (GFP) transgene into a specific locus on the Z chromosome of chicken primordial germ cells (PGCs), resulting in the production of Z^GFP-knockin chickens. By mating Z^GFP-knockin females (Z^GFP/W) with wild males (Z/Z) and using a GFP detection system, we could identify chick sex, as the GFP transgene was expressed on the Z chromosome only in male offspring (Z^GFP/Z) even before hatching. Conclusion: Our results demonstrate that the CRISPR/Cas9 technical platform with chicken PGCs facilitates the production of specific genome-edited chickens for basic research as well as practical applications.

• Journal of Microbiology and Biotechnology
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• v.29 no.11
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• pp.1790-1798
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• 2019

Flock House virus (FHV), an insect RNA virus, has a bipartite genome. FHV RNA1 can be packaged in turnip yellow mosaic virus (TYMV) as long as the FHV RNA has a TYMV sequence at the 3'-end. The encapsidated FHV RNA1 has four additional nucleotides at the 5'-end. We investigated whether the recombinant FHV RNA1 could replicate in mammalian cells. To address this issue, we prepared in vitro transcribed FHV RNAs that mimicked the recombinant FHV RNA1, and introduced them into baby hamster kidney (BHK) cells. The result showed that the recombinant FHV RNA1 was capable of replication. An eGFP gene inserted into the frame with B2 gene of the FHV RNA1 was also successfully expressed. We also observed that eGFP expression at the protein level was strong at 28℃ but weak at 30℃. Sequence analysis showed that the 3'-ends of the RNA1 and RNA3 replication products were identical to those of the authentic FHV RNAs. This indicates that FHV replicase correctly recognized an internally-located replication signal. In contrast, the 5'-ends of recombinant FHV RNA1 frequently had deletions, indicating random initiation of (+)-strand synthesis.

• Proceedings of the KOSOMBE Conference
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• v.1997 no.11
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• pp.475-477
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• 1997

We have analyzed the fluorescence image obtaining from green fluorescence protein (GFP). In order to monitor the fluorescence of specific gene, we used the amyloid precursor protein promoter which has been known to act as a major role in the development of Alzheimer's disease. The promoter from - 3.0 kb to + 100 base pair was inserted into the gene expression monitoring GFP vector purchased from Clontech. This construct was transfected into the PC 12 and fibroblast cells and the fluorescence image was captured by two kinds of methods. One is using cheaper CCD camera and other is SIT-CCD camera. or the higher sensitivity of the fluorescence image, we developed the multiple image grabbing program. As a results, the fluorescence image by conventional CCD camera have the similar sensitivity compared with that of the SIT-camera by applying the multiple image grabbing programs. By this system. it will be possible to construct the fluorescence monitoring system with lower cost. And gene expression in real time by fluorescence image will be possible without changing the fluorescence images.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 1998.05a
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• pp.12-28
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• 1998

The technology of creating transgenic animals has a potential value in improving productivity and disease resistance of animals, gene therapy, drug pharming and production of model animals for certain diseases. Up to date, fairly low success rate of production of transgenic animals and a pronounced variability with respect to the expression of transgenes have been much observed. The mechanisms how to integrate the injected genes with a certain part of the genomes are unknown yet. Many techniques in gene transfer, beside microinjection, have been introduced and explored thus to improve the production efficiency of transgenic animals. In this article, the methods and efficiency of gene-transfer techniques, the detection and preselection of transgenes in embryos by PCR- and GFP-screenings and cloning of preselected transgenic embryos by nuclear transplantation are described and discussed. Some experimental results showed that the early screening and selection of integration of the injected gene with embryonic genome by polymerase chain reaction(PCR) and green fluorecence protein(GFP) were promising methods. Further, the application of nuclear transplantation technology to cloning and multiplication of the positively integrated genes in the cleaving embryos and embryonic cells will be beneficially used for the mass production of transgenic embryos and consequently improving the production efficiency in transgenic animals.

• International Journal of Industrial Entomology and Biomaterials
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• v.1 no.1
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• pp.19-23
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• 2000

A novel recombinant baculovirus Autographa californica nuclear polyhedrosis virus (ACNPV) producing the green fluorescent polyhedra was constructed and characterized. The recombinant virus was stably produced fluorescent polyhedra in the infected cells and the morphology of the polyhedra was nearly similar to that of wild-type AcNPV. For the production of the fluorescent polyhedral the green fluorescent protein (GFP) gene was introduced under the control of polyhedrin gene promoter of AcNPV by translational fusion in the front and back of intact polyhedrin gene. The recombinant baculovirus was named as CXEP, As expected, the 93 kDa fusion protein was expressed in the CXEP-infected cells. Interestingly, however, the cells infected with CXEP also showed a 33 kDa protein band as cells infected with wild-type AcNPV. The results of Southern blot analysis and plaque assay suggested that two types of baculoviruses expressing the GFP fusion protein or only native polyhedrin were formed through homologous recombination between two polyhedrin genes in the same orientation. Thus, this system can be applied for the production of recombinant polyhedra with foreign gene product of diverse interest.

• Investigative Magnetic Resonance Imaging
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• v.16 no.1
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• pp.47-54
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• 2012

Purpose : This study was performed to evaluate the characteristics of rat mesenchymal stem cells (RMSCs) transduced with human ferritin gene and investigate $in$ $vitro$ MRI detectability of ferritin-transduced RMSCs. Materials and Methods: The RMSCs expressing both myc-tagged human ferritin heavy chain subunit (myc-FTH) and green fluorescence protein (GFP) were transduced with lentiviurs. Transduced cells were sorted by GFP expression using a fluorescence-activated cell sorter. Myc-FTH and GFP expression in transduced cells were detected by immunofluorescence staining. The cell proliferative ability and viability were assessed by MTT assay. The RMSC surface markers (CD29+/CD45-) were analyzed by flow cytometry. The intracellular iron amount was measured spectrophotometically and the presence of ferritin-iron accumulation was detected by Prussian blue staining. $In$ $vitro$ magnetic resonance imaging (MRI) study of cell phantoms was done on 9.4 T MR scanner to evaluate the feasibility of imaging the ferritin-transduced RMSCs. Results: The myc-FTH and GFP genes were stably transduced into RMSCs. No significant differences were observed in terms of biologic properties in transduced RMSCs compared with non-transduced RMSCs. Ferritin-transduced RMSCs exhibited increased iron accumulation ability and showed significantly lower $T_2$ relaxation time than non-transduced RMSCs. Conclusion: Ferritin gene as MR reporter gene could be used for non-invasive tracking and visualization of therapeutic mesenchymal stem cells by MRI.