• KSBB Journal
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• v.19 no.4
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• pp.327-330
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• 2004

An inducible expression system of poly[(R)-3-hydroxybutyrate] (PHB) depolymerization was established in metabolically engineered Escherichia coli with the PHB biosynthesis genes. The Ralstonia eutropha PHB depolymerase gene was cloned in a vector system containing the PHB biosynthesis genes and expressed under inducible promoter. Recombinant E. coli harboring the PHB biosynthesis genes and depolymerase gene was first cultured for the accumulation of PHB, and then the depolymerase was expressed resulting in the degradation of accumulated PHB into (R)-3-hydroxybutyric acid (R3HB). R3HB could be produced with the concentration of 7.6 g/L in flask culture. Two different PHB biosynthesis genes from Alcaligenes latus and R. eutropha were compared for the production of R3HB. This strategy can be used for the production of enantiomerically pure (R)-hydroxycarboxylic acids with high concentration.

• Reproductive and Developmental Biology
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• v.29 no.1
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• pp.49-55
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• 2005

We cloned cDNA of the PGH(porcine growth hormone) gene and constructed retrovirus vector designed to express PGH gene under the regulation of CMV (cytomegalovirus) promoter. To maximize the expression, WPRE(woodchuck hepatitis virus posttranscriptional regulatory element) sequence was placed at the downstream of the PGH gene. After infection with recombinant viruses, approximately 1×10/sup 6/ PFF(porcine fetal fibroblast) cells released PGH protein into the media as much as 1,400 ng. In a subsequent experiment, a modifications of the retrovirus vector was made to express the PGH gene in a teracycline-inducible manner. In PFF cells carrying these viral vector sequences, addition of doxycycline to the media resulted in 2∼6 fold increase in PGH synthesis. In the modified retrovirus vectors, the WPRE sequence also played a role in boosting the effect of the tetracycline induction. This result indicates that our tetracycline-inducible expression system might be a promising candidate in alleviating the complicate physiological problems caused by constitutive expression of the exogenous genes in the transgenic animals.

• Journal of Microbiology and Biotechnology
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• v.30 no.8
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• pp.1124-1131
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• 2020

Techniques used for the regulation of gene expression facilitate studies of gene function and treatment of diseases via gene therapy. Many tools have been developed for the regulation of gene expression in mammalian cells. The Lac operon system induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) is one of the employed inducible systems. IPTG mimics the molecular structure of allolactose and has a strong affinity for the corresponding repressor. IPTG is known to rapidly penetrate into mammalian cells and exhibits low toxicity. In the present study, we developed a new inducible expression system that could regulate the expression of genes in mammalian cells using IPTG. Here we confirm that unlike other vector systems based on the Lac operon, this expression system allows regulation of gene expression with lactose in the mammalian cells upon transfection. The co-treatment with IPTG and lactose could improve the regulatory efficiency of the specific target gene expression. The regulation of gene expression with lactose has several benefits. Lactose is safe in humans as compared to other chemical substances and is easily available, making this technique very cost-effective.

• Reproductive and Developmental Biology
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• v.29 no.1
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• pp.57-62
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• 2005

One of the critical problems to be solved in transgenic animal production is uncontrollable constitutive expression of foreign genes, which usually results in serious physiological disturbances in the transgenic animal. To circumvent this problem, we constructed and tested two retrovirus vectors designed to express the GFP(green fluorescent protein) gene under the control of the tetracycline-inducible promoters. To maximize the GFP gene expression at turn-on state, WPRE(woodchuck hepatitis virus posttranscriptional regulatory element) sequence was introduced into the retrovirus vectors at downstream region of either the GFP gene or the sequence encoding rtTA(reverse tetracycline-controlled transactivator). Transformed cells were cultured in the medium supplemented with or without doxycycline(tetracycline derivative) for 48 hours, and induction efficiency was measured by comparing the GFP gene expression level using fluorometry and western blotting. Higher GFP expression was observed from the vector carrying the WPRE sequence at 3' side of the GFP gene, while tighter expression control(up to 20 fold) was obtained from the vector in which the WPRE sequence was placed at 3' side of rtTA sequence. The resulting tetracycline inducible vector system may be used in transgenic animal production and gene therapy.

• Reproductive and Developmental Biology
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• v.30 no.3
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• pp.157-162
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• 2006

Endogenous 84 amino acid parathyroid hormone(PTH) is synthesized as a pre-pro hormone by the chief cells of the parathyroid glands. Physiological actions of PTH include regulation of bone metabolism, renal tubular reabsorption of calcium and phosphate, and intestinal calcium absorption. In addition, PTH stimulates new bone formation by extraordinary stimulation of osteoblastic activity and decreasing calcium excretion by the kidney. In this study, we constructed and tested retrovirus vectors designed to express the human parathyroid hormone(hPTH) gene under the control of the tetracycline-inducible promoters. To increase the hPTH gene expression at turn-on state, woodchuck hepatitis virus posttranscriptional regulatory element(WPRE) sequence was also introduced into retrovirus vector at downstream region of either the hPTH gene or the sequence encoding reverse tetracycline-controlled transactivator(rtTA). Transformed primary culture cells(porcine fetal fibroblast, PFF, chicken embryonic fibroblast, CEF) were cultured in the medium supplemented with or without doxycycline(tetracycline derivative) for 48 hours, and induction efficiency was measured by comparing the hPTH gene expression level using two step RT-PCR and ELISA Higher hPTH expression($3{\tims}10^4\;pg/ml,\;5.3{\times}10^4\;pg/ml$) and tighter expression control(up to 8 fold) were observed from the vector in which the WPRE sequence was placed at downstream of the hPTH gene. The resulting tetracycline inducible vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals.

• Journal of Microbiology and Biotechnology
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• v.18 no.4
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• pp.639-647
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• 2008

The heat-inducible expression vectors for Corynebacterium glutamicum and C. ammoniagenes were constructed by using the ${\lambda}O_L1$ and the cryptic promoters, CJ1 and CJ4 that express genes constitutively in C. ammoniagenes. Although the promoters were isolated from C. ammoniagenes, CJ1 and CJ4 were also active in C. glutamicum. To construct vectors, the $O_L1$ from the ${\lambda}P_L$ promoter was isolated and fused to the CJ1 and CJ4 promoters by recombinant PCR. The resulting artificial promoters, CJ1O and CJ4O, which have one ${\lambda}O_L1$, and CJ1OX2, which has two successive ${\lambda}O_L1$, were fused to the green fluorescent protein (GFP) gene followed by subcloning into pCES208. The expression of GFP in the corynebacteria harboring the vectors was regulated successfully by the temperature-sensitive cI857 repressor. Among them, C. ammoniagenes harboring plasmid pCJ1OX2G containing GFP fused to CJ1OX2 showed more GFP than the other ones and the expression was tightly regulated by the repressor. To construct the generally applicable expression vector using the plasmid pCJ1OX2G, the His-tag, enterokinase (EK) moiety, and the MCS were inserted in front of the GFP gene. Using the vector, the expression of pyrR from C. glutamicum was tried by temperature shift-up. The results indicated that the constructed vectors (pCeHEMG) can be successfully used in the expression and regulation of foreign genes in corynebacteria.

• Reproductive and Developmental Biology
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• v.40 no.1
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• pp.7-13
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• 2016

Effectiveness of transgene transfer into genome is crucially concerned in mass production of the bio-pharmaceuticals using genetically modified transgenic animals as a bioreactor. Recently, the mammary gland has been considered as a potential bioreactor for the mass production of the bio-pharmaceuticals, which appears to be capable of appropriate post-translational modifications of recombinant proteins. The mammary gland tissue specific vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals. In this study, to minimize physiological disturbance caused by constitutive over-expression of the exogenous gene, we constructed new retrovirus vector system designed for mammary gland-specific expression of the hEPO gene. Using piggyBac vector system, we designed to express hEPO gene under the control of mammary gland tissue specific and lactogenic hormonal inducible goat ${\beta}$-casein or mouse Whey Acidic Protein (mWAP) promoter. Inducible expression of the hEPO gene was confirmed using RT-PCR and ELISA in the mouse mammary gland cells treated with lactogenic hormone. We expect the vector system may optimize production efficiency of transgenic animal and reduce the risk of global expression of transgene.

• Archives of Pharmacal Research
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• v.12 no.3
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• pp.176-180
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• 1989

Four bacterial strains having inducible resistance to erythromycin were isolated from soil samples in Korea and characterized. MLS inducibility was checked in each strain. Cloning of inducible resistance gene(s) has been tried. Four isolates were identified as B. anthracis, Micrococcus luteus, Streptococcus faecium and B. licheniformis, in which erythromycin, oleandomycin, cirramycin and carbomycin acted as resistance inducers respectively. The resistance gene cloned from B, licheniformis 597 strain using pBS 42 vector was found to have a 3.2 kb insert.

• Korean Journal of Microbiology
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• v.29 no.6
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• pp.392-396
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• 1991

Mu d1(Ap lac) bacteriophage can be used to search for genes which are members of a common regulatory network without having to know the functions of the genes in advance. Aim was for obtaining the loci in the SOS network as well as temperature inducible loci. For this purpose, recA441 allele was used. This allele encodes a thermosensitive recA gene product; thus, the recA441 allele can be activated upon temperature upshift without by external DNA damage. Approximately 10, 000 colonies were screened, and then searched for the colonies which expressed .betha.-galactosidase higher level at 42.deg.C than at 30.deg.C. The strains identified fell into two dlasses; (i) ones in which the increased expression was $recA^{+}$ $lexA^{+}$ -dependent, that is, din(damage-inducible) genes which were due to the activation of recA441 allele and (ii) ones in which the increased expression was $recA^{+}$ $lexA^{+}$ -independent and only temperature-inducible, tin genes. Rough mapping position was obtained for these genes.

• Parasites, Hosts and Diseases
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• v.53 no.6
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• pp.777-783
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• 2015

The nitric oxide (NO) formation and intrinsic nitrosation may be involved in the possible mechanisms of liver fluke-associated carcinogenesis. We still do not know much about the responses of inducible NO synthase (iNOS) induced by Clonorchis sinensis infection. This study was conducted to explore the pathological lesions and iNOS expressions in the liver of mice with different infection intensity levels of C. sinensis. Extensive periductal inflammatory cell infiltration, bile duct hyperplasia, and fibrosis were commonly observed during the infection. The different pathological responses in liver tissues strongly correlated with the infection intensity of C. sinensis. Massive acute spotty necrosis occurred in the liver parenchyma after a severe infection. The iNOS activity in liver tissues increased, and iNOS-expressing cells with morphological differences were observed after a moderate or severe infection. The iNOS-expressing cells in liver tissues had multiple origins.