• Journal of Sericultural and Entomological Science
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• v.39 no.1
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• pp.36-43
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• 1997

In order to express the FLP recombinase in B. mori cultured cell line, BmN-4, transient expression system using a heat shock protein gene (hsp70) promoter of Dorosophilla melnogaster was constructed. This vector was designated as pHsSV. Activity strength of the hsp70 promoter was compared with that of immediate early gene (IE-1) and polyhedrin gene of BmNPV employing the E. coli $\beta$-galactosidase gene as a reporter gene. The result showed that the pHs $\beta$-gal plasmid vector expressed the $\beta$-galactosidase at 2nd and 3rd day after the transfer of plasmid DNA into BmN-4 cells, which was similar to that of pIE1 $\beta$-gal vector, but different from that of a recombinant virus, vBm $\beta$-gal. For the construction of FLP recombinase transient expression vector, the FLP recombinase gene was cloned by polymerase chain reaction technique. To express the FLP recombinase, this gene was inserted into pHsSV plasmid vector, under the control of the hsp70 promotor, and tranfected in BmN-4 cells. The expressed FLP recombinase was estimated at 44kDa on a 12.5% SDS-PAGE.

• Korean Journal of Ecology and Environment
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• v.52 no.4
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• pp.394-402
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• 2019

The Agrobacterium tumefaciens mediated gene transfer is widely used to generate genetic transformation of plants and transient assay of temporal exogenous gene expression. Syringe infiltration system into tobacco (Nicotiana benthamiana) leaves is a powerful tool for transient expression of target protein to study protein localization, protein-protein binding and protein production. However, the protocol and technical information of transient gene expression, especially double strand RNA (dsRNA), in tobacco using Agrobacterium is not well known. Recently, dsRNA is crucial for insecticidal effect on destructive agronomic pest such as Corn rootworm. In this study, we investigated the factor influencing the dsRNA expression efficiency of syringe agro-infiltration in tobacco. To search the best combination for dsRNA transient expression in tobacco, applied two Agrobacterium cell lines and three plant vector systems. The efficiency of dsRNA expression has estimated by real-time PCR and digital PCR. As a result, pHellsgate12 vector constructs showed the most effective accumulation of dsRNA in the cell. These results indicated that the efficiency of dsRNA expression was depending on the kind of vector rather than Agrobacterium cells. In summary, the optimized combination of transient dsRNA expression system in tobacco might be useful to in vivo dsRNA expression for functional study and risk assessment of dsRNA.

• Microbiology and Biotechnology Letters
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• v.29 no.1
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• pp.18-24
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• 2001

Expression of the baculovirus major envelope glycoprotein gene(gp64) is regulated by transcription from botha early and late promoters. To develop a transient expression vector under the control of gp64 gene promoter, the gp64 gene of Bombyx mori nucleopolyhedrovirus-K1(BmNPV-K1) was characterized. The gp64 gene was local-ized at EcoR I-Pst I 7.38-kb fragment of the BmNPV-K1 genome. The EcorR 1-Pst I 7.38-kb fragment was cloned and the nucleotide sequence of 2,277 bases including the coding region of gp64 gene was determined. Based on these results, transient expression vector using gp64 gene promoter was constructed and named as pBm64. E.coli lacZ gene was introduced onto pBm64 as a reporter gene and expressed transiently in B. mori 5(Bm 5) cells. The expression vector transfected into the cells was maintained stably for 1 to 5 days. In order to confirm the expression of the reporter gene by gp64 promoter, recombinant virus was constructed. The recombinant virus has two independent transcription units in opposite orientations with two promoters; gp64 and polyhedrin gene promoters each initiating transcription of $\beta$-galactosidase and polyhedrin, respectively. Polyhedra formation and expression of $\beta$-galactosidase in Bm5 cells infected with the recombinant virus were observed with phase contrast microscope and in situ staining.

• Journal of Aquaculture
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• v.11 no.4
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• pp.457-463
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• 1998

An expression vector, pUC19N6-luc, containing nuclear matrix attachment region(MAR) isolated from Misgurnus mizolepis liver and control expressino vector, pUC19-luc, were constructed. After these vectors were transferred into CHSE-214 cell line by electroporation, the expression rate of luckferase gens, copy number of vectors and chromosome integration of vectors were analyzed by using assay of luciferase activity, PCR and Southern blotting. While the expression pattern of luciferase gene of pUC19-luc was shown in typicla transient ecpression pattern, that of pUC19N6-luc was highly increased at the 5 days after transfectrion. Although the cope number of pUC19N6-luc vector was higher than that of pUC19-luc vector, these vectors were integrated into chromosome at the same time point in the transfected CHSE-214 cells. In conclusion, the increase of luciferase gene expression of pUC19N6-luc was resulted from not the maintaining of the high copy number but the formation of transcription-favorable structure by MAR effect after chromosomal integration.

• Journal of Plant Biology
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• v.37 no.1
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• pp.77-83
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• 1994

DNA uptake in dry embryos of rice by DNA imbibition was detected by monitoring the expression of chimeric vectors. The selective markers of expression vectors used were ${\beta}-glucuronidase$ ronidase (GUS) and hygromycin phosphotransferase (HPT) genes under the control of CaMV35 S promoter. Frequency of transient expression of the foreign gene was generally 30-50% varying according to the types of vectors and rice cultivars. Dot blot analysis and DNA sequence analysis of inverse polymerase chain reaction products showed that selected rice in hygromycin B (HmB) medium had HPT gene and CaMV35S promoter DNA sequence in genomic DNA of rice. To investigate what ratio of rice having two marker genes simultaneously as rice embryos imbibed the vector DNA having two HPT and GUS gene, transform ants selected in lImB medium were subjected to PCR for GUS gene. It was shown that about 90 percentage of surviving ones in HmB medium had GUS gene.S gene.

• The Journal of Korean Society of Virology
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• v.29 no.1
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• pp.33-38
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• 1999

Retroviral vector provide a highly efficient method for gene transfer into eukaryotic cells. This vector system can be divided into two components; the retroviral vector itself and the retroviral packaging cell line. The key improvement in the design of these two components are, focused on two aspects; the reduction of helper virus production and high titer-virus. We used PA317 for retrovirus packaging cell line, for its high producibility of viral titer. To test the ability of the vectors to generate high titer-virus, we have chosen four different retroviral vectors; LN, LNSX, LNCX and LXSN. To test easily the viral titer, we have made recombinant construction with CD4 and CD8, checked their viral titer and stained their surface expression. LXSN which contain SV40 early promoter in front of neo gene showed best results in viral transient transfection assay, dot blot assay and surface expression. In addition, recombinant containing CD8 generally showed much higher viral titration and surface expression than CD4.

• Journal of Microbiology and Biotechnology
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• v.10 no.1
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• pp.91-94
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• 2000

The suspension cells of Taxus cuspidata were transformed with Agrobacterium tumefaciens harboring binary vector pCAMBIE1302 encoding mgfp. Transient transfection efficiency was compared by using the fluoremetric measurement. The transient transfection efficiency was improved by transformation with DMSO and/or sonication treatment. Optimum conditions for DMSO and sonication treatment were 3% and 30sec, respectively. selection and maintenance of transformed cells were continued for 3 months. An insertion of the mgfp gene in transformed cells was detected by PCR and an expression of GFP confirmed by the western blot analysis.

• Journal of Microbiology and Biotechnology
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• v.22 no.2
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• pp.190-198
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• 2012

RNA interference (RNAi) is rapidly becoming a valuable tool in biological studies, as it allows the selective and transient knockdown of protein expression. The short-interfering RNAs (siRNAs) transiently silence gene expression. By contrast, the expressed short-hairpin RNAs induce long-term, stable knockdown of their target gene. Trichoplusia ni (T. ni) cells are widely used for mammalian cell-derived glycoprotein expression using the baculovirus system. However, a suitable shRNA expression system has not been developed yet. We investigated the potency of shRNA-mediated gene expression inhibition using human and Drosophila U6 promoters in T. ni cells. Luciferase, EGFP, and ${\beta}$-N-acetylglucosaminidase (GlcNAcase) were employed as targets to investigate knockdown of specific genes in T. ni cells. Introduction of the shRNA expression vector under the control of human U6 or Drosophila U6 promoter into T. ni cells exhibited the reduced level of luciferase, EGFP, and ${\beta}$-N-acetylglucosaminidase compared with that of untransfected cells. The shRNA was expressed and processed to siRNA in our vector-transfected T. ni cells. GlcNAcase mRNA levels were down-regulated in T. ni cells transfected with shRNA vectors-targeted GlcNAcase as compared with the control vector-treated cells. It implied that our shRNA expression vectors using human and Drosophila U6 promoters were applied in T. ni cells for the specific gene knockdown.

• Journal of Photoscience
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• v.9 no.2
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• pp.225-228
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• 2002

Bcl-2 is a member of large bcl-2 family and protects cells from apoptosis. Using bcl-2-expressing adenovirus vector (Ad-bc1-2), we investigated the effect of bc1-2 on UVB-induced apoptosis. Adenovirus vector efficiently introduced bc1-2 gene in cultured normal mouse keratinocytes (NMK cells); almost all NMK cells (lx10$^{6}$ ) were transfected at Ixl0$^{8}$ PFU/ml. Bcl-2-transfected NMK cells were significantly resistant to UVB-induced apoptosis with the suppressive effect dependent on bcl-2-expression level. Following UVB irradiation caspase 8, 3, 9 activities were stimulated in NMK cells, while in bc1-2-transfected cells, only caspase 8, but not caspase 3 or 9 activities were stimulated. In order to investigate the effect of bc1-2 in vivo, topical application of Ad-bc1-2 on tape-stripped mouse skin was performed. Following the application, Bc1-2 was efficiently overexpressed in almost all viable keratinocytes. The expression was transient with the maximal expression of Bc1-2 at 1st day following the application of lxl0$^{9}$ PFU in 200ml. The introduced Bc1-2 remained at least for 6 days. UVB irradiation (1250 J/m$^2$) induced apoptosis within 12 h and the maximal effect was observed at 24 h in control mouse skin. Bc1-2-transfected mice skin were resistant to UVB-induced apoptosis. Topical application of empty adenovirus vector alone had no effect on Bc1-2 expression or UVB-induced apoptosis. These results indicate that adenovirus vector is an efficient gene delivery system into keratinocytes and that Bcl-2 is a potent inhibitor of UVB-induced apoptosis both in vitro and in vivo.

• Proceedings of the Korean Society of Applied Entomolohy Conference
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• 2001.05a
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• pp.110-110
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• 2001