• Proceedings of the Korean Society of Toxicology Conference
• /
• 2000.11a
• /
• pp.45-67
• /
• 2000

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a mutagenic and carcinogenic heterocyclic amino found in cooked meat. The in vivo mutagenicity and hepatocarcinogenicity of MeIQx were examined in mice harboring the lacZ mutation reporter gene ($Muta^{TM}$ Mice) and bitransgenic mice over-expressing the c-myc oncogene. C57B1/$\lambda$lacZ and bitransgenic c-myc (albumin promoter)/$\lambda$lacZ mice were bred and weaned onto an AIN-76 based diet containing $0.06\%$ (w/w) MeIQx or onto control diet. After 30 weeks on diet, only male bitransgenic mice on MeIQx developed hepatocellular carcinoma ($100\%$ incidence) indicating that there was synergism between c-myc over-expression and MeIQx. By 40 weeks, hepatic tumor incidence was $100\%$ ($17\%$) and $44\%$ ($0\%$) in male c-myc/$\lambda$lacZ and C57B1/$\lambda$lacZ mice given MeIQx (or control) diet, respectively, indicating that either MeIQx or c-myc over-expression alone eventually induced hepatic tumors. At either time point, mutant frequency in the lacZ gene was at least 40-fold higher in MeIQx-treated mice than in control mice of either strain. These findings suggest that MeIQx-induced hepatocarcinogenesis is associated with MeIQx-induced mutations. Elevated mutant frequency in MeIQx-treated mice also occurred concomitant with the formation of MeIQx-guanine adducts as detected by the $^{32}P$-postlabeling assay. Irrespective of strain or diet, sequence analysis of the lacZ mutants from male mouse liver showed that the principal sequence alteration was a single guanine-base substitution. Adenine mutations, however, were detected only in animals on control diet. MeIQx-fed mice harboring the c-myc oncogene showed a l.4-2.6-fold higher mutant frequency in the lacZ gene than mice not carrying the transgene. Although there was a trend toward higher adduct levels in c-myc mice, MeIQx-DNA adduct levels were not significantly different between c-myc/$\lambda$lacZ and C57B1/$\lambda$lacZ mice after 30 weeks on diet. Thus, it appeared that factors in addition to MeIQx-DNA adduct levels, such as the enhance rate of proliferation associated with c-myc over-expression, may have accounted for a higher mutant frequency in c-myc mice. In the control diet groups, the lacZ mutant frequency was significantly higher in c-myc/$\lambda$lacZ mice than in 057B1/$\lambda$1acZ mice. The findings are consistent with the notion that c-myc over-expression is associated with an increase in mutagenesis. The mechanism for the synergistic effects of c-myc over-expression on MeIQx hepatocarcinogenicity appears to involve an enhancement of MeIQx-induced mutations.

• Korean Journal of Plant Tissue Culture
• /
• v.22 no.3
• /
• pp.175-182
• /
• 1995

Rice is one of the most successful monocot in regenerating fertile and genetically stable transgenic plants. However there is no report of a rice line developed in Korea that can be used for regeneration of fertile and genetically stable transformants. In this paper we first demonstrate that a Korean variety Nakdongbyeo, is suitable to obtain transgenic rice plants. Protoplasts from embryogenic suspension cultures were co-transformed with HPT (hygromycin phosphotransferase) and GUS ($\beta$-glucuronidase) genes in separate plasmids in the presence of PEG (polyethylene glycol). In 5 independent experiment, the average frequency of calli showing hygromycin resistance were 1.73%. Plantlets were regenerated from the Hy $g^{R}$ calli. The average efficiency of plantlet regeneration was apprbximately 27%. Based on the GUS activities of hygromycin resistant calli, ca.35% of the resistant calli carried active GUS genes. The R0 transgenic plantlets were grown to maturity and Rl seeds were obtained. By examining the in siぉ activity of GUS in Rl seeds and seedlings, we confirmed that the GUS transgene driven by a CaMV 35S (cauliflower mosaic virus) promoter showed proper expression patterns. We also confirmed Mendelian segregation of the HPT transgene in the Rl generation.n.

• Biomedical Science Letters
• /
• v.11 no.2
• /
• pp.165-173
• /
• 2005

A potent demethylating agent, 5-Azacytidine (5-AzaC) has been widely used as in many studies on DNA methylation, regulation of gene expression, and cancer biology. The mechanisms of the demethylating activity were known to be formation of complex between DNA and DNA methyltransferase (MTase), which depletes cellular MTase activity. However, 5-AzaC can also induce hypermethylation of a transgene in a transgenic cell line, G12 cells and it was explained as a result of defense mechanisms to inactivate foreign gene(s) somehow. This finding evoked the question that whether the phenomenon of hypermethylation induced by 5-AzaC is limited to the transgene or it can be occurred in endogenous gene(s). In order to answer the question, mutagenicity test of 5-AzaC and molecular characterization of mutants obtained from the test were performed using an endogenous gene, thymidine kinase (tk) in Chinese hamster V79 cells. When V79 and V79-J3 subclone cells were treated with 1, 2.5 ,5, $10{\mu}M$ of 5-AzaC for 48 hours, their maximum mutant frequencies were revealed as $6\times10^{-3}\;at\;5{\mu}M$(350-fold induction over background) and $8\times10^{-3}\;at\;2.5{\mu}M$ (l,800-fold induction over background) respectively. Since the induction rates were too high to be induced by true mutations, many trifluorothymidine (TFT)-resistant $(TFT^R)$ cells were subjected to Northern blot analysis to check the presence of tk transcripts. Surprisingly, all clones tested possessed the transcripts in a similar level, that implicates the $TFT^R$ phenotype induced by 5-AzaC has not given rise to hypermethylation of the gene in spite of unusually high mutation frequency. In addition, it has shown that the TK activity in the pool of 5-AzaC-induced $TFT^R$ cells has about a half of that in spontaneously-induced $TFT^R$ cells or in non-selected parental V79-J3 cells. This result suggests that the mechanism(s) underlying the TFT-resistance between spontaneously occurred and 5-AzaC-induced cells may be different. These findings have shown that the $TFT^R$ phenotype induced by 5-AzaC has not given rise to hypermethylation of the tk gene, and 5-AzaC may be induced by one or combined pathways among many drug resistance mechanisms. The exact mechanisms for the 5-AzaC-induced $TFT^R$ phenotype remain to elucidate.

• Korean Journal of Breeding Science
• /
• v.42 no.5
• /
• pp.540-546
• /
• 2010

This study was conducted to understand the role of soybean pathogenesis-related 10 (GmPR10) gene in salt tolerance and to develop salt-tolerant rice using GmPR10 cDNA. GmPR10 transgene was expressed constitutively in the shoot and root of the $T_1$ transgenic rice plants. Interestingly, however, the levels of the transgene expression were increased temporally up to over four- to five-fold in the shoot and root by 125 mM NaCl treatment, peaking at six hours after the treatment and decreasing thereafter. Electrolyte leakage of leaf cells under 125 mM NaCl treatment was lower in all the transgenic lines than in the control variety, Dongjin-byeo. Ability of seedlings to recover from 125 mM NaCl treatment for two weeks was higher in the transgenic plants than in the control plants. These results demonstrated that GmPR10 had function to increase cell integrity and promote growth under the saline stress imposed by NaCl. The transgenic line GmPR10-3 which showed highest ability to recover from the saline stress could be used as a potential source for salt tolerance in rice breeding programs.

• Horticultural Science & Technology
• /
• v.31 no.1
• /
• pp.95-102
• /
• 2013

Binary trans-activation (pOp/LhG4) system is one of the regulatory systems of transgene expression. The target gene expression is achieved by crossing the reporter plants with an activator in this system. In this study, we used the features of this system in Chinese cabbage as a way to protect genetic resources and new varieties. To establish pOp/LhG4 system in Chinese cabbage, we designed an activator (35SLhG41300), and reporter constructs (pOpGUSBart) and co-transformed using Agrobacterium. The transgenic plants were selected by antibiotics and the functional activity of pOp/LhG4 system was confirmed by GUS expression. To induce the tissue-specific function, we constructed pOp/LhG4 system (795LhGBart) using female tissue specific promoter (ProAt1g26795) of Arabidopsis. Co-transformed transgenic plants clearly showed tissue specific expression in Arabidopsis. The results suggest the possibility of the system's application of $F_1$ generation can be restricted by expressing the target gene to protect a new variety and genetic resource in Chinese cabbages.

• Horticultural Science & Technology
• /
• v.18 no.5
• /
• pp.594-598
• /
• 2000

This study was carried out to investigate the tissue-specific expression of ${\beta}$-glucuronidase (gus) gene driven by endosperm-specific promoter (Z4 promoter) in the transgenic tobacco and to find out inheritance pattern of transgene to the next generation. Tobacco (Nicotiana tabaccum cv. Havana SR1) was transformed with Agrobacterium tumerfaciens LBA4404 harboring BV3 construct containing gus gene driven by Z4 promoter and a kanamycin resistant gene. Seven hundred bp PCR products, indicating the presence of npt II gene, were found in the all eight transformants by PCR analysis using nptII primers. To study the expression pattern of the two different kind of promoters, leaf disks of the Z4pro-gus-transformed plants and 35Spro-gus-transformed plants were analyzed histochemically for gus activity. As a result, leaf disks of Z4pro-gus-transformed plants showed very weak and partial positive gus activity. In contrast, leaf disks of 35Spro-gus-transformed plants showed relatively strong positive gus activity. To investigate the expressed position of Z4 promoter, seeds from Z4pro-gus-transformed plants and 35Spro-gus-transformed plants were analyzed histochemically for gus activity. Z4pro-gus-transformed seeds showed positive gus activity restricted to the endosperm. However, the blue-colored product in 35Spro-gus-transformed seeds was observed in all the area including endosperm. Kanamycin resistance assay showed that transgenes were stably inherited to next generation in all lines.

• Korean Journal of Animal Reproduction
• /
• v.24 no.4
• /
• pp.439-449
• /
• 2000

This study was to investigate the expression of transgene after co-injection of spermatozoon and EGFP gene into mature oocytes in cattle. From frozen semen, spermatozoa were treated by DTT with 0.03% Tween-20, freezing and thawing or 0.02% Triton X-100 to disrupt their plasma membranes. The sperm injected oocytes were co-cultured with bovine oviduct epithelial cells in CRlaa, and expression of EGFP in embryos were observbed under epifluorescent microscope. Two pronuclei were formed in oocytes injected with sperm treated by DTT (44.6%), DTT-Tween-20 (48.4%), DTT-freezing (44.4%) and DTT-Triton X-100 (42.9%). Cleavage and blastocyst formation rates of bovine oocytes which injected with sperm treated by DTT, DTT-Tween-20, DTT-freezing, and DTT-Triton X-100 were 49.1, 58.5, 43.9, and 48.4% and 10.2, 13.0, 6.8, and 6.5%, respectively. Although the most of embryos were showing mosaicism, embryos expressing EGFP gene were observed in all treated groups. Therefore, these results indicate that membrane disrupted sperm could interact with exogenous DNA, and that this procedure may be useful to introduce foreign gene into bovine oocytes.

• Korean Journal of Poultry Science
• /
• v.36 no.2
• /
• pp.177-186
• /
• 2009

It is well-known that unregulated over-expression of foreign gene may have unwanted physiological or toxic effects in transgenic animals. To circumvent these problems, we constructed retrovirus vector designed to express the foreign gene under the control of the tetracycline-inducible promoter. However, gene expressions in the tetracycline-inducible expression system (Tet system) are not completely regulated but a little leaky due to the inherent defects in conventional Tet-based systems. A more tightly controllable regulatory system can be achieved when the advanced versions ($rtTA2^SM2$) of rtTA and a minimal promoter in responsive components (pTRE-tight) are used in combination therein. In this study, we tried to produce human thrombopoietin (hTPO) from various target cells and transgenic chickens using the retrovirus vector combined with Tet system. hTPO is the primary regulator of platelet production and has an important role in the survival and expansion of hematopoietic stem cells. In a preliminary experiment in vitro, higher hTPO expression and tighter expression control were observed in chicken embryonic fibroblast (CEF) cells. We also measured the biological activity of the hTPO using Mo7e cells whose proliferation is dependant on hTPO. The biological activity of the recombinant hTPO from CEF was higher than both its commercial counterpart and hTPO from other target cells. The recombinant retrovirus was injected beneath the blastoderm of non-incubated chicken embryos (stage X). Out of 138 injected eggs, 15 chicks hatched after 21 days of incubation. Among them, 8 hatched chicks were hTPO positive. When the Go transgenic chicken was fed doxycycline (0.5 mg per 1 gram of feed), a tetracycline derivative, hTPO concentration of the transgenic chicken blood was 200 ng/mL. Germline transmission of the transgene was confirmed in sperm of the Go transgenic roosters. These results are informative to establish transgenic chickens as bioreactors for the mass production of commercially valuable and biological active human cytokine proteins.

• Korean Journal of Microbiology
• /
• v.47 no.1
• /
• pp.1-6
• /
• 2011

Inefficiency of in vivo gene transfer using currently available vectors reflects a major hurdle in cancer gene therapy. Both viral and non-viral approaches have been described to improve gene transfer efficiency but suffer from a number of limitations. Here we tested an adenovirus carrying the small peptide ligand derived from heregulin${\beta}$ EGF-like domain onto fiber, the adenoviral capsid protein, to deliver transgene to ovarian cancer cells which overexpress ErbB, the cognate receptors for heregulin. The attachement of 53 amino acids to fiber didn't affect on the fiber's trimer structure that is critical for the viral entry to cells. The fiber-modified adenovirus can mediate entry and expression of a ${\beta}$-galactosidase into cancer cells in an increased efficiency compared the unmodified adenovirus. Particularly, the gene transfer efficiency was improved up to 5 times in OVCAR3 cells, an ovarian cancer cell line. Such transduction systems hold promise for delivering genes to ErbB receptor overexpressing cancer cells, and could be used for future cancer gene therapy.

• Journal of Ecology and Environment
• /
• v.36 no.1
• /
• pp.39-47
• /
• 2013

The expression of transgenic traits in genetically modified crops is sometimes associated with decreases in crop performance or fitness. These decreases in performance or fitness of transgenic plants in unfavourable conditions may provide valuable information about the ecological consequences of transgene escape. In a glasshouse trial, we tested the cost associated with resistance to herbicides by comparing the growth, yield, and competitive ability of transgenic rice with its parental non-transgenic line. This new line was developed for constitutive overexpression of protoporphyrinogen oxidase (PPO) to increase resistance to herbicides. We evaluated nine agronomic traits of transgenic and non-transgenic rice grown in a replacement series design over four densities. Competitive ability was also assessed between transgenic and non-transgenic plants by analyzing their relative yields based on biomass and seed weight data. Our results indicated that non-transgenic plants showed greater performance than did the transgenic plants when those genotypes were grown in mixtures. The non-transgenic rice plants exhibited superior competitive ability at certain combinations of planting densities and genotype proportions. These results suggest that PPO-herbicide resistance incurs some costs in plant performance and competitive ability.