• Clinical and Experimental Pediatrics
• /
• v.51 no.2
• /
• pp.170-180
• /
• 2008

Purpose : Some antibiotics were known to exert neuroprotective effects in the animal model of hypoxic-ischemic (H-I) brain injury, but the mechanism is still unclear. A recent study reported that geneticin (G418), an aminoglycoside antibiotic, increased survival of human breast cancer cells by suppressing apoptosis. We investigated the neuroprotective effects of systemically administrated geneticin via anti-apoptosis following the H-I brain injury Methods : Seven-day-old Sprague-Dawley rat pups were subjected to unilateral (left) common carotid artery occlusion followed by 2.5 hours of hypoxic exposure and the cortical cell culture of rat brain was done under a hypoxic incubator. Apoptosis was measured in the injured hemispheres 7 days after H-I insult and in the injured cells from hypoxic chamber using morphologic analysis by Terminal dUTP Nick-end Labeling(TUNEL) assay and immunohistochemistry for caspase-3, and cytologic analysis by western blot and real time PCR for bax, bcl-2, and caspase-3. Results : The gross appearance and hematoxylin and eosin stain revealed increased brain volume in the geneticin-treated animal model of perinatal H-I brain injury. The TUNEL assay revealed decreased apoptotic cells after administration of geneticin in the cell culture model of anoxia. Immunohistochemistry showed decreased caspase-3 expression in geneticin-treated cortical cell culture. Western blot and real-time PCR showed decreased caspase-3 expression and decreased ratio of Bax/Bcl-2 expression in geneticin-treated animal model. Conclusion : Geneticin appears to exert a neuroprotective effect against perinatal H-I brain injury at least via anti-apoptosis. However, more experiments are needed in order to demonstrate the usefulness of geneticin as a preventive and rescue treatment for H-I brain injuries of neonatal brain.

• Journal of Life Science
• /
• v.24 no.10
• /
• pp.1046-1054
• /
• 2014

Beta-1,3-glucanase is widely used in various biotechnological and industrial processes, with over-production required to enable versatile utilization. We examined the overexpression of ${\beta}$-1,3-glucanase (EXGA) from Aspergillus oryzae using ${\delta}$-sequence-mediated integration. We constructed $pRS{\delta}$-exgA and $pRS{\delta}K$-exgA plasmids for integration of the EXGA gene into various chromosomes of Saccharomyces cerevisiae. These plasmids contain the ADH1 promoter for constitutive expression, a signal sequence (exoinulinase signal sequence [INU1 s.s]) for secretory production, and a ${\delta}$-sequence for integration of ${\beta}$-1,3-glucanase. The $pRS{\delta}$-exgA plasmid was transformed into the S. cerevisiae $BY4742{\Delta}exg1$ strain, and ${\beta}$-1.3-glucanase was stably overexpressed and secreted. Another plasmid, $pRS{\delta}K$-exgA, was introduced into the S. cerevisiae $BY4742{\Delta}exg1$ (YKY082) strain, and overexpression of ${\beta}$-1,3-glucanase was examined by inducible integration under geneticin selection. The activity of ${\beta}$-1,3-glucanase increased in accordance with a rise in the geneticin concentration, with 0.8 mg/ml of geneticin suitable for overexpression of ${\beta}$-1,3-glucanase. Subsequently, $pRS{\delta}K$-exgA was repeatedly transformed for sequential ${\delta}$-integration. The activity of ${\beta}$-1,3-glucanase reached about 0.063 unit/ml/$OD_{600}$, 0.095 unit/ml/$OD_{600}$, 0.131 unit/ml/$OD_{600}$ and 0.165 unit/ml/$OD_{600}$ by the first, second, third, and fourth round of integration, respectively. According to the increase in the activity of ${\beta}$-1,3-glucanase by sequential ${\delta}$-integration, the copy number (integration rate) of the EXGA gene also increased in various chromosomes. These results suggest that recombinant ${\beta}$-1,3-glucanase activity can be sequentially increased by repeated ${\delta}$-sequence integration.

• Parasites, Hosts and Diseases
• /
• v.49 no.4
• /
• pp.357-364
• /
• 2011

Various Leishmania species were engineered with green fluorescent protein (GFP) using episomal vectors that encoded an antibiotic resistance gene, such as aminoglycoside geneticin sulphate (G418). Most reports of GFP-Leishmania have used the flagellated extracellular promastigote, the stage of parasite detected in the midgut of the sandfly vector; fewer studies have been performed with amastigotes, the stage of parasite detected in mammals. In this study, comparisons were made regarding the efficiency for in vitro G418 selection of GFP-Leishmania amazonensis promastigotes and amastigotes and the use of in vivo G418 selection. The GFP-promastigotes retained episomal plasmid for a prolonged period and G418 treatment was necessary and efficient for in vitro selection. In contrast, GFP-amastigotes showed low retention of the episomal plasmid in the absence of G418 selection and low sensitivity to antibiotics in vitro. The use of protocols for G418 selection using infected BALB/c mice also indicated low sensitivity to antibiotics against amastigotes in cutaneous lesions.

• Journal of Plant Biotechnology
• /
• v.2 no.3
• /
• pp.143-149
• /
• 2000

An Efficient in vitro regeneration system and an optimized Agrobacterium mediated transformation protocol are described, based on the use of young seedling cotyledons of Capsicum annuum L. Optimal regeneration efficiency can be obtained by cultivating cotyledon explants on media containing 4 mg/L benzyladenine and 0.1 mg/L indolacetic acid. The effect of antibiotics used to eliminate Agrobacteria, as well as the toxic level of some generally used selection agents (kanamycin, geneticin, hygromycin, phosphinotricin and methotrexate) in regenerating pepper tissues were determined. To enable the comparison of different selection markers in identical vector background, a set of binary vectors containing the marker genes for NPTII, HPT, DHFR and BAR respectively, as well as the CaMV 35S promoter/enhancer-GUS chimaeric gene was constructed and introduced into four different Agrobacterium host strains.

• Journal of Plant Biotechnology
• /
• v.7 no.4
• /
• pp.241-246
• /
• 2005

Factors affecting regeneration of different Rubus varieties (blackberry, raspberry and their hybrid) were examined and a reliable regeneration system was established. Media for stock plant maintenance were tested; different explants and media were investigated to find the best circumstances for the regeneration. The effect of the commonly used antibiotics was studied to determine the most suitable one for selection of the transformants. We found that both MS and LS media supplemented by $20\;gL^{-1}$ sucrose are suitable for the stock plant maintenance. The optimal hormone content for the stock plants is $0.125\;mgL^{-1}$ 6-benzylaminopurine (BAP) with $0.01\;mgL^{-1}$ indole-3- butyric acid (IBA). The highest regeneration rate was observed on medium containing MS salts with B5 vitamins complemented with glucose, sucrose, maltose, $10\;gL^{-1}$ each, supplemented with benzylaminopurine riboside (BAR) ($2\;mgL^{-1}$) and indole-3-acetic acid (IAA) ($0.1\;mgL^{-1}$). The regenerated shoots appeared directly from the cut edges, without callus phase. Hygromycin and geneticin proved to be good selection agents for the Rubus explants, but due to their severe effect on the tissues we propose to use marker-free constructions for the transformation.

• Journal of Microbiology and Biotechnology
• /
• v.26 no.3
• /
• pp.477-482
• /
• 2016

A uridine diphosphate-glucose:sterol glycosyltransferase-encoding gene was isolated and cloned from the established fosmid library of Micromonospora rhodorangea ATCC 27932 that usually produces the aminoglycoside antibiotic geneticin. The gene consists of 1,185 base pairs and encodes a 41.4 kDa protein, which was heterologously expressed in Escherichia coli BL21(DE3). In silico analyses of the deduced gene product suggested that it is a member of the family 1 glycosyltransferases. The recombinant protein MrSGT was able to catalyze the transfer of a glucosyl moiety onto the C-3 hydroxy function in sterols (β-sitosterol, campesterol, and cholesterol), resulting in the corresponding steryl glucosides (β-sitosterol-3-O-β-ᴅ-glucoside, campesterol-3-O-β-ᴅ-glucoside, and cholesterol-3-O-β-ᴅ-glucoside). This enzyme prefers phytosterols to cholesterol, and also shows substrate flexibility to some extent, in that it could recognize a number of acceptor substrates.

• Journal of Microbiology and Biotechnology
• /
• v.9 no.5
• /
• pp.668-671
• /
• 1999

The gene encoding Schwanniomyces occidentalis $\alpha-amylase$(AMY) was introduced into Saccharomyces cerevisiae var. diastaticus which secreted only glucoamylase, by using a linearized yeast integrating vector to develop stable strains with a capability of secreting $\alpha-amylase$and glucoamylase simultaneously. A dominant selectable marker, the geneticin(G418) resistance gene (Gt^r$), was cloned into a vector to screen wild-type diploid transformants harboring the AMY gene. The amylolytic activities of transformants were about 3-7 times higher than those of the recipient strains. When grown in nonselective media, the transformants with the linearized integrating vector containing the AMY gene exhibited almost all of the mitotic stability after 100 generations.

• Journal of Microbiology and Biotechnology
• /
• v.11 no.1
• /
• pp.65-71
• /
• 2001

A gene from Paenibacillus sp. KCTC 8848P encoding ${\beta}-amylase$ was cloned and expressed in Escherichia coli. The Paenibacillus ${\beta}-amylase$ gene cosisted of a 2,409-bp open reading frame without a translational stop codon, encoding a protein of 803 amino acids. The presumed ribosime-binding site, GGAGG, was located 10 bp upstream from the TTG initiation codon. The deduced amino acid sequence of the ${\beta}-amylase$ gene had a 95% similarity to the ${\beta}-amylase$ of Bacillus firmus. The ${\beta}-amylase$ gene was introduced into wild-type strains of Saccharomyces cerevisiae using a linearized yeast integrating vector containing a geneticin resistance gene and its product was secreted into the culture medium.

• Proceedings of the KSAR Conference
• /
• 2003.06a
• /
• pp.55-55
• /
• 2003

The random insertion of useful gene in genome has been a common method to produce transgenic animals. This method is inefficient for induction of high levels gene expression in transgenic animals. To improve this limit, we tried to develop the system which target the gene at the specific genomic region. Thus, in our experiment, the vector system to target the human thrombopoietin (TPO) gene was developed. Targeting vector including TPO, neo and DT genes was transfrcted into bovine embryonic fibroblasts (bEF) or bovine ear skin fibroblasts (bESF). First of all, we determined concentration of the geneticin (G418) for selection of transfected cell lines. Our results showed that 1200 and 900 $\mu\textrm{g}$/ml of G418 were the most proper for selection of transfscted bEF and bESF cells. In this study, lipofectamine was used as a transfection reagent. Thus, the proper ratio of DNA:lipofectamine for transfection was also required to elevate targeting efficiency in primary mammalian cells. Our result indicates that the most proper ratios of DNA:lipofectamine were 4:2 and 1:2 in bEF and bESF cells. According to the optimized these conditions, single colonies were picked following transfection and were analyzed by PCR. More than 90% of the single colonies have TPO gene. However, there were no colonies with targeted TPO at the specific genomic region. Therefore, further experiments to select the specifically targeted colonies and to find more efficient methods such as reducing selection time and shortening a size of TPO gene are required.

• Journal of Microbiology and Biotechnology
• /
• v.25 no.11
• /
• pp.1856-1862
• /
• 2015

In order to improve the stability of endoglucanase under thermal and acidic conditions, the endoglucanase gene was fused to the N-terminus of the Saccharomyces cerevisiae pir gene, encoding the cell wall protein PIR. The fusion gene was transformed into Pichia pastoris GS115 for expression. A resulting strain with high expression and high activity was identified by examining resistance to Geneticin 418, Congo red staining, and quantitative analysis of enzyme activity. SDS-PAGE analysis revealed that the endoglucanase was successfully displayed on the yeast cell surface. The displayed endoglucanase (DEG) showed maximum activity towards sodium carboxyl methyl cellulose at approximately 275 IU/g cell dry weight. DEG exhibited greater than 60% residual activity in the pH range 2.5-8.5, higher than free endoglucanase (FEG), which had 40% residual activity at the same pH range. The highest tolerated temperature for DEG was 70℃, much higher than that of FEG, which was approximately 50℃. Moreover, DEG showed 91.1% activity at 65℃ for 120 min, while FEG only kept 77.8% residual activity over the same period. The half-life of DEG was 270 min at 65℃, compared with only 150 min for FEG. DEG could be used repeatedly at least three times. These results suggest that the DEG has broad applications as a yeast whole-cell biocatalyst, due to its novel properties of high catalytic efficiency, acid-thermal stabilities, and reusability.