• Journal of Applied Biological Chemistry
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• v.53 no.4
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• pp.189-194
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• 2010

We confirmed the hypo-osmotic shock strengths and duration, different type of vectors, and subcelluar localization to identify the optimum analysis condition of plant aquaporin activity in Xenopus ooctye using Arabidopsis thaliana AtPIP2-1 gene. Six minutes and 1/5ND buffer hypoosmotic shock treatment was the best condition to show the maximum swelling of Xenopus oocytes where AtPIP2-1 was expressed using pcDNA3.1 vector. AtPIP2-1 protein was expressed more efficiently in pGEMHE vector which has 5' and 3' UTR (untranslation region) of Xenopus ${\beta}$-GLOBIN gene in multiple cloning site than in pcDNA3.1 vector. Also green fluorescence of GFP fused to AtPIP2-1 was detected onto oocyte plasmamembrane where is the proper subcellular localization target of AtPIP2-1.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.6
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• pp.2555-2559
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• 2015

Purpose: To investigate effects of the TESTIN (TES) gene on proliferation and migration of highly metastatic nasopharyngeal carcinoma cell line 5-8F and the related mechanisms. Materials and Methods: The target gene of human nasopharyngeal carcinoma cell line 5-8F was amplified by PCR and cloned into the empty plasmid pEGFP-N1 to construct a eukaryotic expression vector pEGFP-N1-TES. This was then transfected into 5-8F cells. MTT assays, flow cytometry and scratch wound tests were used to detect the proliferation and migration of transfected 5-8F cells. Results: A cell model with stable and high expression of TES gene was successfully established. MTT assays showed that the OD value of 5-8F/TES cells was markedly lower than that of 5-8F/GFP cells and 5-8F cells (p<0.05). Flow cytometry showed that the apoptosis rate of 5-8F/TES cells was prominently increased compared with 5-8F/GFP cells and 5-8F cells (p<0.05). In vitro scratch wound assays showed that, the width of the wound area of 5-8F/TES cells narrowed slightly, while the width of the wound area of 5-8F/ GFP cells and 5-8F cells narrowed sharply, suggesting that the TES overexpression could inhibit the migration ability. Conclusions: TES gene expression remarkably inhibits the proliferation of human nasopharyngeal carcinoma cell line 5-8F and reduces its migration in vitro. Thus, it may be a potential tumor suppressor gene for nasopharyngeal carcinoma.

• KSBB Journal
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• v.21 no.6 s.101
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• pp.461-465
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• 2006

We investigated the protein productivity of the promoters for genes showing prolonged induction upon cold shock in Escherichia coli. Six low temperature inducible genes (frdA, glpB, hypB, katG, nupG, ompT) were selected based on the previously reported cDNA microarray based global transcription profiling of Escherichia coli Kl2 in response to cold shock. Their promoter regions were isolated from the genomic DNA of E. coli JM109 and expression levels induced by the promoters were examined by using green fluorescence protein (GFP) as a reporter at $15^{\circ}C$ and $37^{\circ}C$. Among the six promoters, the promoter for nupG showed the highest and prolonged expression at both temperatures and the cold inducibility of nupG promoter was not observed.

• BMB Reports
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• v.53 no.3
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• pp.160-165
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• 2020

The root meristem of Arabidopsis thaliana is protected by the root cap, the size of which is tightly regulated by the balance between the formative cell divisions and the dispersal of the outermost cells. We isolated an enhancer-tagged dominant mutant displaying the short and twisted root by the overexpression of ZINC-FINGER OF ARABIDOPSIS THALIANA1 (ZAT1) encoding an EAR motif-containing zinc-finger protein. The growth inhibition by ZAT1 was shared by ZAT4 and ZAT9, the ZAT1 homologues. The ZAT1 promoter was specifically active in the outermost cells of the root cap, in which ZAT1-GFP was localized when expressed by the ZAT1 promoter. The outermost cell-specific expression pattern of ZAT1 was not altered in the sombrero (smb) or smb bearskin1 (brn1) brn2 accumulating additional root-cap layers. In contrast, ZAT4-GFP and ZAT9-GFP fusion proteins were distributed to the inner root-cap cells in addition to the outermost cells where ZAT4 and ZAT9 promoters were active. Overexpression of ZAT1 induced the ectopic expression of PUTATIVE ASPARTIC PROTEASE3 involved in the programmed cell death. The EAR motif was essential for the growth inhibition by ZAT1. These results suggest that the three related ZATs might regulate the maturation of the outermost cells of the root cap.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.3
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• pp.781-784
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• 2012

The aim of this study was to evaluate the effect of the adenovirus-mediated double suicide gene (CD/TK) for selective killing of gastric cancer cells. Gastric cancer cells SCG7901 and normal gastric epithelial cell lines were infected by adenoviruses Ad-survivin/GFP and Ad-survivin/CD/TK. GFP expression and CD-TK were detected by fluorescence microscopy and reverse transcriptase polymerase chain reaction (RT-PCR), respectively. After treatment of the infected cells with the pro-drugs ganciclovir (GCV) and/or 5-FC, the cell growth status was evaluated by methyl thiazolyl tetrazolium assay. Cell cycle changes were detected using flow cytometry. In nude mice bearing human gastric cancer, the recombinant adenovirus vector was injected directly into the tumor followed by an intraperitoneal injection of GCV and/or 5-FC. The subsequent tumor growth was then observed. The GFP gene driven by survivin could be expressed within the gastric cancer line SCG7901, but not in normal gastric epithelial cells. RT-PCR demonstrated the presence of the CD/TK gene product in the infected SCG7901 cells, but not in the infected normal gastric epithelial cells. The infected gastric cancer SCG7901, but not the gastric cells, was highly sensitive to the pro-drugs. The CD/TK fusion gene system showed significantly greater efficiency than either of the single suicide genes in killing the target cells (P<0.01). Treatment of the infected cells with the pro-drugs resulted in increased cell percentage in G0-Gl phase and decreased percentage in S phase. In nude mice bearing SCG7901 cells, treatment with the double suicide gene system significantly inhibited tumor growth, showing much stronger effects than either of the single suicide genes (P<0.01). The adenovirus-mediated CD/TK double suicide gene driven by survivin promoter combined with GCV an 5-FC treatment could be an effective therapy against experimental gastric cancer with much greater efficacy than the single suicide gene CD/TK combined with GCV or 5-FC.

• Journal of Microbiology and Biotechnology
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• v.24 no.1
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• pp.70-79
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• 2014

In an attempt to develop a variety of expression vector systems for Corynebacterium glutamicum, six types of promoters, including $P_{tac}$, $P_{sod}$, $P_{sod}$ with a conserved Shine-Dalgarno (SD) sequence from C. glutamicum, $P_{ilvC}$, $P_{ilvC}$ with a conserved SD-1 ($P_{ilvC-M1}$), and $P_{ilvC}$ with a conserved SD-2 ($P_{ilvC-M2}$), were cloned into a modified shuttle vector, pCXM48. According to analysis of promoter strength by quantitative reverse transcription PCR, $P_{sod}$ and $P_{sod-M}$ were superior to tac and ilvC promoters in terms of transcription activity in C. glutamicum. All of the promoters have promoter activities in Escherichia coli, and $P_{sod-M}$ displayed the highest level of transcriptional activity. The protein expression in constructed vectors was evaluated by measuring the fluorescence of green fluorescent protein (GFP) and SDS-PAGE. C. glutamicum harboring plasmids showed GFP fluorescence with an order of activity of $P_{ilvC}$ > $P_{ilvC-M1}$ > $P_{sod}$ > $P_{ilvC-M2}$ > $P_{sod-M}$, whereas all plasmids except pCSP30 with $P_{sod}$ displayed fluorescence activities in E. coli. Of them, the strongest level of GFP was observed in E. coli with $P_{sod-M}$, and this seems to be due to the introduction of the conserved SD sequence in the translational initiation region. These results demonstrate that the expression vectors work well in both C. glutamicum and E. coli for the expression of target proteins. In addition, the vector systems harboring various promoters with different strengths, conserved SD sequences, and multiple cloning sites will provide a comfortable method for cloning and gene expression, and consequently contribute to the metabolic engineering of C. glutamicum.

• Korean Journal of Microbiology
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• v.45 no.4
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• pp.300-305
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• 2009

We constructed the deletion mutants of fission yeast Schizosaccharomyces pombe spNab2 gene that is homologous to poly(A)-binding protein NAB2 in budding yeast Saccharomyces cerevisiae, which plays crucial roles in mRNA 3' end formation and mRNA export from nucleus into the cytoplasm. A null mutant in an $h^+$/ $h^+$ diploid strain was constructed by replacing the spNab2-coding region with an $ura4^+$ gene using one-step gene disruption method. Tetrad analysis showed that the spNab2 is not essential for vegetative growth and mRNA export. However, over-expression of spNab2 cause the severe growth defects and intensive accumulation of poly(A) RNA in the nucleus. Also, the spNab2-GFP fusions were localized mainly in the nucleus. These results suggest that spNab2 is also involved in mRNA export out of the nucleus.

• Genomics & Informatics
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• v.7 no.4
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• pp.203-207
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• 2009

The target of rapamycin (TOR) signaling pathway conserved from yeast to human plays critical roles in regulation of eukaryotic cell growth. It has been shown that TOR pathway is involved in several cellular processes, including ribosome biogenesis, nutrient response, autophagy and aging. However, due to the functional diversity of TOR pathway, we do not know yet some key effectors of the pathway. To find unknown effectors of TOR signaling pathway, we took advantage of a green fluorescent protein (GFP)-tagged collection of budding yeast Saccharomyces cerevisiae. We analyzed protein abundance changes by measuring the GFP fluorescence intensity of 4156 GFP-tagged yeast strains under inhibition of TOR pathway. Our proteomic analysis argues that 83 proteins are decreased whereas 32 proteins are increased by treatment of rapamycin, a specific inhibitor of TOR complex 1 (TORC1). We found that, among the 115 proteins that show significant changes in protein abundance under rapamycin treatment, 37 proteins also show expression changes in the mRNA levels by more than 2-fold under the same condition. We suggest that the 115 proteins indentified in this study may be directly or indirectly involved in TOR signaling and can serve as candidates for further investigation of the effectors of TOR pathway.

• Journal of Embryo Transfer
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• v.30 no.3
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• pp.225-228
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• 2015

Understanding the behavior of transgenes introduced into oocyte or embryos is essential for evaluating the methodologies for transgenic animal production. To date, many studies have reported the production of transgenic pig embryos with, however, low efficiency in environment of blastocyst production. The aim of present study was to determine the expression and duration of transgene transferred by intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT). Embryos obtained from the ICSI-MGT procedure were analysed for the expression of GFP and then for the transmission of the transgene. Briefly, fresh spermatozoa were bound to exogenous DNA after treatment by Triton X-100 and Lipofectin. When ICSI-MGT was performed using sperm heads with tails removed, the yield of blastocyst (25.3%), treated with Lipofectin (18.8%) and Triton X-100 (19.2%) were observed. Treatments of Lipofectin or Triton X-100 did not further improve the rates of blastocysts. Moreover, the apoptosis rates of embryos were obtained from the control and LIpofectin groups (8.7%, 9.7%, respectively), but were significantly higher in the Triton X-100 group (13.0%). Our results demonstrated that ICSI-MGT caused minimal damage to oocytes that could develop to full term. Moreover, the embryos derived by ICSI-MGT have shown prolonged exogenous DNA expression during preimplantation stage in vivo. However, more efforts will be required to improve the procedures of both sperm treatments cause of high frequency of mosaicisms.

• Korean Journal of Poultry Science
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• v.27 no.2
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• pp.155-161
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• 2000

Unfortunately, there is no technique which is stable and repetitive to produce transgenic chicken, although various ways of gene transfer including PGC-and embryonic cell-mediated gene transfer, DNA microinjection, virus inoculation and sperm cells have been employed. The aims of this study were 세 develop and establish such a stable, repetitive and efficient way of gene transfer giving a faithful gene expression during development after the reconstruction of embryo in an UV-irradiated egg. A dual reporter plasmid (pJJ9), a fusion gene containing lacZ and GFP driven by a CMV promoter was used to exploit either merits of both reporting markers. lacZ with strong signal or GFP with vital marking. Electroporated embryonic blastodermal cells (EBCs) in the presence of the pJJ9 DNA faithfully showed 377 bp PCR product and lacZ or GFP expressions in the identical cells in vitro of in vivo. Furthermore, analyses of expression pattern of the foreign DNA demonstrated that microinjected EBCs cells into the UV-irradiated recipient egg should participate in normal developmental process, for example, proliferation and differentiation into various tissues. Thirty percentages of the manipulated eggs showed lacZ expression in their tissues. These results together with the specific procedures used in this study should facilitate avian transgenesis.