• Journal of Plant Biotechnology
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• v.6 no.1
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• pp.9-13
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• 2004

Transient uidA expression was used to optimize parameters required for biolistic transformation of suspension cells of Easter lily, Lilium longiflourm. Maximum uidA expression occurred following bombardment with gold particles as compared to tungsten. A 3hr pre-treatment of suspension cells with 0.125M osmoticum resulted in a 1.5X increase in uidA expression. A helium pressure of 1550 psi combined with a particle travelling distance of 6cm resulted in maximum uidA expression as compared to either 1100, 1200, or 1800 psi. Transient transformation resulted in up to 493 uidA expressing cells/Petri plate. For stable transformation suspension cells of Lilium longiflorum, were co-bombarded with plasmid DNA containing cucumber mosaic virus (CMV) replicase under the rice actin (Act1) promoter and either the bar or PAT genes under the cauliflower mosaic virus (CaMV 355) promoter. Ten regenerated plants contained the transgene as analyzed by PCR, and two of the ten plants were confirmed to contain the transgene by Southern hybridization. The two transgenic plants were independent transformants, one containing the bar gene and the other both the CMV replicase and bar genes. Plants were sprayed at the rosette stage and found to be resistant to 1000 mg/L of phosphinothricin (Trade name-Ignite) indicating expression of the bar gene throughout the leaves when bar was under control of the CaMV 35S promoter.

• Journal of Plant Biotechnology
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• v.37 no.3
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• pp.319-326
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• 2010

An interest in the production of seed-oil based fuel and raw materials, which comes from renewable plant sources, has been intrigued by the phenomenon of global warming and shortage of fossil fuels. Rapeseed (Brassica napus) is the most important oilseed crop, which produces seeds with 40% oil. It is desirable to develop genetically modified rapeseed producing oils, which can be easily converted to biodiesel. As an initial step for development of genetically modified rapeseed for the production of biofuels or bio-based materials, Korean rapeseed cultivars, Naehan, Youngsan, Tammi and Halla, were analyzed. Four Korean rapeseed cultivars produce 32 to 40% oil of seed dry weight, which is rich in oleic acid (more than 60 mole%). The cotyledonary petioles of rapeseed cultivar, Halla, were transformed using Agrobacterium tumefaciens strain GV3101, carrying the uidA gene encoding $\beta$-glucuronidase (GUS) as a reporter gene and the phosphinothricin acetyltransferase (PAT) gene as a selectable marker. The stable integration of PAT gene in the genome of transgenic rapeseeds was confirmed by PCR analysis. Expression of uidA gene in various rapeseed organs was determined by fluorometric assay and histochemical staining. Transformation efficiency of a Korean rapeseed Halla cultivar was 10.4%. Genetic inheritance of transgenes was confirmed in $T_2$ generation.

• Tuberculosis and Respiratory Diseases
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• v.53 no.2
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• pp.113-126
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• 2002

Background : DNA repair plays a crucial role in protection from cancer-causing agents. Therefore, a reduced DNA repair capacity can increase the susceptibility to lung cancer. The XPC gene contains 15 exons and encodes a 940 amino acid protein that plays a central role in DNA damage recognition of the nucleotide excision repair pathway, which is a major DNA repair mechanisim removing the bulky-helix distorting DNA lesions caused by smoking. Recently several polymorphisms in the XPC gene were identified. In addition, it is possible that these polymorphisms may affect the DNA repair capacity, which modulate cancer susceptibility. The relationship between codon 499 and 939 polymorphisms, and a poly(AT) insertion/deletion polymorphism in the XPC gene, and the lung cancer risk were investigated. Materials and Methods : The genotypes were determined using either PCR or PCR-RFLP analysis in 219 male lung cancer patients and 150 healthy males controls. Results : The frequencies of the genotypes (Val499Ala, PAT and Lys939Gln) among the cases were not significantly different from those of the controls. There was no significant associantion between these polymorphi는 and the lung cancer risk when the analyses were stratified according to age, smoking status and the pack-years of smoking. Moreover, the genotypes had no apparent relationship with any of the histological types of lung cancer. There was a linkage disequilibrium among the Val499Ala, PAT and Lys939Gln polymorphisms. The PAT polymorphism had a strong linkage disequilibrium with the Lys939Gln polymorphism (kappa value=0.87). The XPC haplotypes showed no significant association with the lung cancer risk. Conclusion : These results suggest that XPC Val499Ala, PAT and Lys939Gln polymorphisms are not major contributors to the individual lung cancer susceptibility in Koreans.

• Reproductive and Developmental Biology
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• v.30 no.2
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• pp.125-133
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• 2006

This study was conducted to detect the apoptosis incidence in blastocysts and to compare the abundance of Bax, Bcl2L1, VEGF and FGFR2 in in vitro fertilized (IVF), parthenogenetic (PAT) and nuclear transfer (NT) embryos. Oocytes matured for 40 hr were enucleated and reconstructed with confluenced fetal fibroblasts (FFs) derived from a ${\sim}45$ day fetus. Reconstructed eggs were then fused with 2 DC pulses (2.0 kV/cm, $30{\mu}sec$) and cultured with $7.5{\mu}g/ml$ cytochalasin B for 3 hr. Parthenotes (PAT) were produced with the same electric strength and culture for NT eggs. The embryos were cultured in NCSU-23 medium at $39^{\circ}C,\;5%\;CO_2,\;5%\l;O_2$ in air. In 3 runs, set of 10 embryos at the 4-cell to blastocyst stages were used to extract total RNA for analyzing the gene expression patterns of pro-apoptotic (Bax), anti-apoptotic (Bcl2L1), vasculogenesis (VEGF), implantation (FGFR2III) using real-time quantitative PCR. Cleavage and blastocyst rates were significantly higher (P<0.05) in IVF and PAT ($79.3{\pm}8.5\;and\;25.5{\pm}6.1,\;and\;85.0{\pm}6.4\;and\;38.6{\pm}5.5$, respectively)than NT counterparts ($65.1{\pm}5.2\;and\;15.6{\pm}3.0$, respectively). Significantly higher (P<0.05) total cells were observed in IVF controls and PAT ($34.7{\pm}5.8\;and\;38.1{\pm}4.1$) than NT embryos ($24.8{\pm}3.2$). Apoptosis index was significantly lower (P<0.05) in IVF than NT embryos. The Relative abundances (RA) of Bax and VEGF were significantly higher (P<0.05) at blastocyst stage in NT than IVF control. The RA of Bcl2L1 and FGFR2III were significantly higher (P<0.05) at blastocyst stage in IVF than NT. The present study observed the abnormal gene expressions in NT embryos at various developmental stages, suggesting certain clues to find out the cause of the low efficiency of NT to term.

• Animal cells and systems
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• v.2 no.2
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• pp.239-242
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• 1998

Arabidopsis trp1 mutant plants, deficient in phosphoribosyI anthranilate transferase (PAT) activity, accumulate anthranilate compounds, which render them blue fluorescence. The visible phenotype of trp1 makes the PAT gene an excellent reporter gene in the mutant. In order to develop a system for the homologous recombination using the phenotypic characteristic of trp1-100, we established optimum conditions for the isolation and regenera tion of protoplast from auxin-conditioned, trp1-100 root cultures. Trvptophan had to be supplemented in the germination medium for the efficient cell division and subsequent plant regeneration. When 10 uM tryptophan was added to the germination medium, we obtained the highest yield of protoplasts ($3{\times}10^6 cells/g$) and the best viability (92%). Thirty percent of root protoplast derived from meristematic cells underwent cell division within 5 days in callus-induction medium. Regenerated rosette leaves (2-3 mm) were transferred to rooting medium and finally acclimated to the soil for flowering.

• Journal of Life Science
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• v.14 no.2
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• pp.368-373
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• 2004

We have obtained fertile transgenic rice plants resistant to the broad spectrum herbicide Bast $a^{(R)}$ (active ingredient phosphinothricin, PPT) by PEG-mediated transformation procedure. The plasmid pCaMV35S::Bar was used to deliver the bar gene into embryogenic suspension culture-derived protoplasts of rice (Oryza sativa L.). Transformed plants were regenerated and selected on medium containing 15 mg/l of phosphinothricin. Stable integration and expression of the bar gene in transgenic rice plants was confirmed by Southern and Northern blot analysis. Transgenic $R_1$ plants were also confirmed by assays for phosphinothricin acetyltransferase (PAT) activity. The bar gene was expressed in the primary transgenic rice plants and in the next generation progeny, in which it showed a 3 : 1 Mendelian inheritance pattern. Transgenic $R_1$ and $R_2$ plants were resistant to the herbicide Bast $a^{(R)}$ when sprayed at rates used in field practice.ice.

• Applied Biological Chemistry
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• v.46 no.4
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• pp.344-347
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• 2003

A method using PCR was developed for the monitoring of genetically modified soybean (GMS) and GMS derived foods utilized in the market. We designed 3 pairs of specific oligonucleotide primers based on epsps and pat inserted in GMS and ferritin gene as internal standards. Template DNAs isolated from soybean and processed foods were used for multiplex PCR with 3 primer sets. PCR, used with specific primer sets for GMS detection, showed the amplified DNA fragments with GMS template DNA. In this study, GMS containing epsps was detected from soy processed foods manufactured before GM food labeling system, however, GMS containing epsps or pat was not detected from soy processed foods manufactured after GM food labeling system.

• Reproductive and Developmental Biology
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• v.32 no.4
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• pp.229-235
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• 2008

Silencing of Dicer1 by siRNA did not inhibit development up to the blastocyst stage, but decreased expression of selected transcription factors, including Oct-4, Sox2 and Nanog, suggesting that Dicer1 gene expression is associated with differentiation processes at the blastocyst stage (Cui et al., 2007). In order to get insights into genes which may be linked with microRNA system, we compared gene expression profiles in Gapdh and Dicer1 siRNA-microinjected blastocysts using the Applied Biosystem microarray technology. Our data showed that 397 and 737 out of 16354 genes were up- and down-regulated, respectively, following siRNA microinjection (p<0.05), including 24 up- and 28 down-regulated transcription factors. Identification of genes that are preferentially expressed at particular Dicer1 knock down embryos provides insights into the complex gene regulatory networks that drive differentiation processes in embryos at blastocyst stage.

• Journal of Life Science
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• v.11 no.4
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• pp.316-320
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• 2001

Sonication tremendously improves the efficiency of Agrobacterium infection by introducing small and uniform fissures and channels throughout the targeted tissue. Using shoot tips of cotton as explants, the effect of sonication treatment and virulence genes in Agrobacterium tumefaciens on transformation efficiency was investigated. The pat gene which encodes resistance to the herbicide, glufosinate, was used as a selectable marker. Transformation efficiency was evaluated on th basis of survival rates of cocultivated shoot tips on selection medium containing 2.5 mg/l gulfosinate-ammonium(ppt) adn 25. mg/l Clavamax. Sonication from 5 to 15 second has a positive effect on shoop tip survival. However, whil virE as well as virG or vir GN54D showed an enhancement in transformation efficiency, virE,. virG resulted in the most significant enhancement. Overall, the combination of additional virG/virE gene and sonication treatment resulted in the most significant increase in transformation efficiency.

• Korean Journal of Environmental Agriculture
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• v.32 no.3
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• pp.231-239
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• 2013

BACKGROUND: The disease resistant (OsCK1) rice was generated by inserting choline kinase (CK1) and phosphinothricin acetyltransferase (PAT) genes isolated from Oriza sativa and Streptomyces hygroscopicus into the genome of rice (Nakdongbyeo). With the potential problems of safety, the non-target organism evaluation is required as an essential element for the environmental risk assessment of genetically modified (GM) crops. In present study, we studied the effects on survival of Misgurnus anguillicaudatus and Cyprinus carpio, commonly used as a model organism in ecotoxicological studies. METHODS AND RESULTS: The M. anguillicaudatus and C. carpio were fed on disease resistant (OsCK1) rice and non-genetically modified (non-GM) rice (Nakdongbyeo) to 0, 10, 100, 1,000 and 5,000 mg/L, as treatment concentration respectively. The OsCK1 rice used for the test was confirmed to have the OsCK1/PAT gene expression by the PCR and ELISA analysis. Feeding test showed that no significant differences in cumulative immobility and abnormal response of M. anguillicaudatus and C. carpio fed on between OsCK1 rice and non-GM rice. The 96hr-$LC_{50}$ values showed no difference between OsCK1 rice (>5,000 mg/L) and non-GM rice (>5,000 mg/L). CONCLUSION(S): The results of this study suggested that there was no significant difference in toxicity for M. anguillicaudatus and C. carpio between OsCK1 rice and non-GM counterparts.