• Journal of Microbiology and Biotechnology
• /
• v.19 no.9
• /
• pp.1070-1076
• /
• 2009

Telomerase reverse transcriptase (TERT), which prolongs the replicative life span of cells, is highly upregulated in 85-90% of human cancers, whereas most normal somatic tissues in humans express limited levels of the telomerase activity. Therefore, TERT has been a potential target for anticancer therapy. Recently, we described a new approach to human cancer gene therapy, which is based on the group I intron of Tetrahymena thermophila. This ribozyme can specifically mediate RNA replacement of human TERT (hTERT) transcript with a new transcript harboring anticancer activity through a trans-splicing reaction, resulting in selective regression of hTERT-positive cancer cells. However, to validate the therapeutic potential of the ribozyme in animal models, ribozymes targeting inherent transcripts of the animal should be developed. In this study, we developed a Tetrahymena-based trans-splicing ribozyme that can specifically target and replace the mouse TERT (mTERT) RNA. This ribozyme can trigger transgene activity not only also in mTERT-expressing cells but hTERT-positive cancer cells. Importantly, the ribozyme could selectively induce activity of the suicide gene, a herpes simplex virus thymidine kinase gene, in cancer cells expressing the TERT RNA and thereby specifically hamper the survival of these cells when treated with ganciclovir. The mTERT-targeting ribozyme will be useful for evaluation of the RNA replacement approach as a cancer gene therapeutic tool in the mouse model with syngeneic tumors.

• Journal of Embryo Transfer
• /
• v.15 no.2
• /
• pp.167-173
• /
• 2000

The principal objective of this study was to clone transgenic embryos in order to improve the efficiency of transgenic animal production by the combination of microinjection and nuclear transplantation techniques. Mature female New Zealand White rabbits were superovulated by eCG and hCG treatments, fllowed by natural mating. Zygotes were collected from the oviducts at 18∼22 h after hCG injection by flushing with D-PBS containing 5% fetal calf serum(FCS). Two to three picoliters of green fluorescent protein(GFP) gene wa microinjected into male pronucleus. The foreign gene-injected zygotes were cultured in TCM-199 or RD medium containing 10% FCS with a monolayer of rabbit oviductal epithelial cells in a 5% CO2 incubator. The morulae expressing GFP gene were selected and their blastomeres were separated for the use of nuclear donor. Following nuclear transplantation of fluorescence-positive morula stage blastomeres, 13 (21.3%) out of 61 fused oocytes developed to blastocyst stage and all of the cloned blastocysts expressed GFP. The results indicate that the screening of transgene in rabbit embryos by GFP detection could be a promisible method for the preselection of transgenic embryos. Also the cloning of preselected transgenic embryos by nuclear transplantatin could be efficiently applied to the multiple production of transgenic animals.

• Journal of Microbiology and Biotechnology
• /
• v.20 no.5
• /
• pp.917-924
• /
• 2010

The practicability of transgenic tobacco engineered to express bacterial native mercuric reductase (MerA), responsible for the transport of $Hg^{2+}$ ions into the cell and their reduction to elemental mercury ($Hg^0$), without any codon modification, for phytoremediation of mercury pollution was evaluated. Transgenic tobacco plants reduce mercury ions to the metallic form; take up metallic mercury through their roots; and evolve the less toxic elemental mercury. Transformed tobacco produced a large amount of merA protein in leaves and showed a relatively higher resistance phenotype to $HgCl_2$ than wild type. Results suggest that the integrated merA gene, encoding mercuric reductase, a key enzyme of the bacterial mer operon, was stably integrated into the tobacco genome and translated to active MerA, which catalyzes the bioconversion of toxic $Hg^{2+}$ to the least toxic elemental $Hg^0$, and suggest that MerA is capable of reducing the $Hg^{2+}$, probably via NADPH as an electron donor. The transgenic tobacco expressing merA volatilized significantly more mercury than wild-type plants. This is first time we are reporting the expression of a bacterial native merA gene via the nuclear genome of Nicotiana tabacum, and enhanced mercury volatilization from tobacco transgenics. The study clearly indicates that transgenic tobacco plants are reasonable candidates for the remediation of mercurycontaminated areas.

• KSBB Journal
• /
• v.31 no.1
• /
• pp.27-32
• /
• 2016

Several CoA transferases from Clostridium beijerinckii, C. perfringens and Klebsiella pneumoniae were examined for biosynthesis of lactate-containing polyhydroxyalkanoates (PHAs) in recombinant Escherichia coli XL1-Blue strain. The CB3819 gene and the CB4543 gene from C. beijerinckii, the pct gene from C. perfringens and the pct gene from K. pneumoniae, which encodes putative CoA transferase gene, respectively, was co-expressed with the Pseudomonas sp. MBEL 6-19 phaC1437 gene encoding engineered Pseudomonas sp. MBEL 6-19 PHA synthase 1 ($PhaC1_{Ps6-19}$) to examine its activity for the construction of key metabolic pathway to produce poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)]. The recombinant E. coli XL1-Blue expressing the phaC1437 gene and CB3819 gene synthesized poly(3-hydroxybutyrate) [P(3HB)] homopolymer to the P(3HB) content of 60.5 wt% when it was cultured in a chemically defined medium containing 20 g/L of glucose and 2 g/L of sodium 3-hydroxybutyrate. Expression of the phaC1437 gene and CB4543 gene in recombinant E. coli XL1-Blue also produced P(3HB) homopolymer to the P(3HB) content of 51.2 wt% in the same culture condition. Expression of the phaC1437 gene and the K. pneumoniae pct gene in recombinant E. coli XL1-Blue could not result in the production of PHAs in the same culture condition. However, the recombinant E. coli XL1-Blue expressing the phaC1437 gene and the C. perfringens gene could produce poly(3-hydroxybutyrate-co-lactate [P(86.4mol%3HB-co-13.7 mol%LA) up to the PHA content of 10.6 wt% in the same culture condition. Newly examined CoA transfereases in this study may be useful for the construction of engineered E. coli strains to produce PHA containing novel monomer such lactate.

• Journal of Microbiology
• /
• v.37 no.2
• /
• pp.90-96
• /
• 1999

The core protein of the hepatitis C virus (HCV) is a multifunctional protein. The HCV core protein was reported to regulate cellular gene expression and transform primary rat embryo fibroblast cells. However, the role of the core protein in the pathogenesis of HCV-associated liver diseases is not well understood. To investigate the functional role of the core protein in cytophathogenicity, we have constructed stable expression systems of full length or truncated HCV core protein lacking the C-terminal hyderophobic domains and established HepG2 cell clones constitutively expressing the core protein. The full length core protein was localized in the cytoplasm and the C-terminal truncated core protein was localized in the nucleus. HepG2 cells expressing nuclear, truncated core protein showed elevated cell death during cultivation compared to untransfected cells and full length core-expressing cells. In the treatment with bleomycin, both cell clones expressing full length or truncated core protein appeared to be more sensitive to blemoycin than the parental HepG2 cells. These results suggest that the core protein may play a role in HCV pathogenesis promoting apoptotic cell death of infected cells.

• BMB Reports
• /
• v.29 no.3
• /
• pp.183-191
• /
• 1996

DNA fragments, homologous to the dTDP-D-glucose 4,6-dehydratase gene, obtained from the genomic DNA of Streptomyces antibioticus $T\ddot{u}99$, a producer of the unusual macrolide antibiotic chlorothricin, were cloned and sequenced. This dehydratase gene was designated as oxil. The coding region of the oxil gene is composed of 987 bp, and analysis of the DNA sequence data reveals sequences for the gene products of 329 amino acids (molecular weight of 36,037). The deduced amino acids are 59% identical to the StrE, dTDP-D-glucose 4,6-dehydratase from the streptomycin pathway. The oxil's function was examined by expressing it in E. coli using the T7 RNA polymerase/promoter system (pRSET) to produce an active fusion protein including a his tag. This enzyme shows specificity of substrate, specific only to dTDP-D-glucose.

• Journal of Microbiology and Biotechnology
• /
• v.8 no.2
• /
• pp.141-145
• /
• 1998

Overproduction of intracellular ${\beta}$-glucosidase was attempted by modifying the promoter region of a ${\beta}$-glucosidase gene cloned from Cellulomonas fimi and expressing it in Bacillus subtilis DB 104. A strong engineered promoter, BJ27UΔ88, was fused to the ${\beta}$-glucosidase gene after removing its native promoter. An effective Shine-Dalgamo sequence (genel0 of phage T7) was inserted between the promoter and the ${\beta}$-glucosidase structural gene. The modified gene was overexpressed in B. subtilis and produced 1121.5 units of ${\beta}$-glucosidase per mg protein which is about $12\%$ of total intracellular protein. Secretion of overproduced intracellular ${\beta}$-glucosidase was attempted by using the signal sequence of the Bacillus endoglucanase gene as well as an in-frame hybrid protein of endoglucanase. The hybrid protein was normally secreted into the culture medium and still retained ${\beta}$-glucosidase activity.

• Journal of Microbiology and Biotechnology
• /
• v.10 no.2
• /
• pp.275-280
• /
• 2000

The inulin fructotransferse (depolymerizing) (IFTase, EC 2.4.1.93) gene of Arthrobacter sp. A-6 was cloned and expressed in Escherichia coli and Bacillus subtilis. The IFTase gene consisted of an ORF of 1.311 nucleotides encoding a polypeptide of 436 amino acids containing a signal peptide of 31 amino acids in the N-terminus. The molecular mass of the IFTase based on the nucleotide sequence was calculated to be 46.116 Da. The recombinant E. coli $DH5{\alpha}$ cells expressing the Arthrobacter sp. A-6 IFTase gene produced most of the IFTase intracelularly. In contrast, the recombinant B. subtilis DB 104 carrying the IFTas gene on a B. subtilis-E. Coli expression vector secreted the IFTase into the culture fluid efficiently.

• BMB Reports
• /
• v.41 no.5
• /
• pp.358-362
• /
• 2008

RNA interference (RNAi) is the process of sequence-specific gene silencing. However, RNAi efficiency still needs to be improved for effective inhibition of target genes. We have developed an effective strategy to express multiple shRNAs (small hairpin RNA) simultaneously using multiple RNA Polymerase III (Pol III) promoters in a single vector. Our data demonstrate that multiple shRNAs expressed from Pol III promoters have a synergistic effect in repressing the target gene. Silencing of endogenous cyclophilin A (CypA) or key HIV viral genes by multiple shRNAs results in significant inhibition of the target gene.

• Journal of Sericultural and Entomological Science
• /
• v.39 no.2
• /
• pp.180-185
• /
• 1997

To develope the novel baculovirus transfer vector, the p10 gene was cloned from the Bombyx mori nuclear polygedrosis virus (BmNPV) vB2 strain isolated from the B. mori larvae of sericultural farms. The novel transfer vector was constructed by using the p10 gene of BmNPV vB2 strain was 210 bp. The TAAG sequence at the -71 bp of upstream from translation initiator ATG and two polyadenylation signal site at the downstream from terminator TAA were also detected in the p10 gene. The 5' and 3' flanking region of the p10 gene amplified by PCR was cloned into pBluescriptII SK(+) and then transfer vector pBm10 was construceted. The 7.9 kb pBm10 was analysed by restriction enzymes and the map was confirmed. In order to determine the expression of foreign gene of pBm10, $\beta$-galactosidase gene was inserted in the SmaI site of foreign gene cloning site of pBm10. The pBm10 containing $\beta$-galactosidase gene was cotranfected wth genomic DNA of BmNPV vB2 into BmN-4 cells. The recombinant baculovirus expressing $\beta$-galactosidase was also produced polygedra in the infected cells. The results indicated that pBm10 is functional, suggesting that in the baculovirus expression vector system, the recombinant virus produced by pBm10 was effective by oral infection for the producing recombinant proteins in in vivo expression.