• Food Science and Biotechnology
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• v.18 no.5
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• pp.1118-1123
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• 2009

Event-specific real-time polymerase chain reaction (PCR) detection method for genetically modified (GM) maize MIR604 was developed based on integration junction sequences between the host plant genome and the integrated transgene. In this study, 2 primer pairs and probes were designed for specific amplification of 100 and 111 bp DNA fragments from the zSSIIb gene (the maize endogenous reference gene) and MIR604. The quantitative method was validated using 3 certified reference materials (CRMs) with levels of 0.1, 1, and 10% MIR604. The method was also assayed with 14 different plants and other GM maize. No amplification signal was observed in real-time PCR assays with any of the species tested other than MIR604 maize. As a result, the bias from the true value and the relative deviation for MIR604 was within the range from 0 to 9%. Precision, expressed as relative standard deviation (RSD), varied from 2.7 to 10% for MIR604. Limits of detections (LODs) of qualitative and quantitative methods were all 0.1%. These results indicated that the event-specific quantitative PCR detection system for MIR604 is accurate and useful.

• Journal of Aquaculture
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• v.13 no.1
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• pp.21-27
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• 2000

The successful gene transfer and transient expression was demonstrated in olive flounder embryos using lipofected sperm. Olive flounder sperm interacted with foregn plasmid DNA encapsidated by positively charged liposome. The maximum plasmid copy number that associated with the sperm was 5 copies/sperm based on the examination of DNA blot assay. The foreign DNA was transferred into fertilized eggs without any adverse effect on fertilization and survival of embryos (P>0.05) and retained in embryos until at least 42 hours with successful expression. The maximal expression was detected in 18 hours after fertilization at 18$^{\cird}C$ and gradually decreased with development of embryo. Most of DNA transferred into embryos persisted extrachromosomally without significant sign of integration or replication.

• Journal of Aquaculture
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• v.16 no.1
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• pp.51-58
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• 2003

The first Indian transgenic fish was generated in 1991 using borrowed constructs from foreign sources. To construct transformation vectors for the indigenous fishes, growth hormone genes of rohu (r-CH), Labeo rohita and catfish, Heteropneustes fossilis were isolated, cloned and sequenced; their fidelity was confirmed in prokaryotic and eukaryotic systems. A vector was constructed with grass carp b-actin promoter driving the expression of r-GH. Rohu eggs are large. fragile and swell 2~3 times. when fertilized. Hence they were amenable only for electroporated sperm-mediated gene transfer. Accordingly, the sperm electroporation technique was standardized to ensure 25% hatchling survival and 37% Presumptive transgenics without suffering any deformity. Southern analysis confirmed genomic integration in 15% of the tested individuals (Ti) belonging to family lines 2 and 3: another 25% of the Juveniles (Te) were also proved transgenic but with the transgene persisting extrachromosomally for longer than 1 to 2 years. perhaps due to the presence of replicon in the vector. Transgenics belonging to different family lines grew 6~8 times faster than the respective controls. Difference in growth trends of Ti and Te within a family line was not significant. In the Ti family 3 remarkable growth acceleration was sustained for a period longer than 36 weeks but in those of family 2, it gradually decreased. All transgenic fishes including the rohu converted the food at a significantly higher efficiency. Barring the transgenic mudloach, all the other transgenic fishes consumed food at significantly reduced rate.

• The Korean Journal of Mycology
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• v.39 no.3
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• pp.235-238
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• 2011

Agrobacterium harboring vector pCHBs with hygromycin phosphotransferase(hph) and hepatitis B virus surface antigen(HBsAg)gene was transformed into the mycelial culture of Flammulina velutipes. In particular, mild NaOH solution was treated to the mycelia before Agrobacterium infection step. This was purposed to generate putative surface wounds in the mycelial cell walls. The results showed that hygromycin-resistant($hyg^r$) mycelia could be obtained only from NaOH-treated mycelia but not from intact mycelia. The integration of $hyg^r$ gene in fungal genome was confirmed by PCR. In addition, a single transgene integration and heterologous protein expression in F. velutipes could be verified by Southern blot hybridization and western blot analysis, respectively. This study demonstrated an efficient tool for the Agrobacterium-mediated transformation of F. velutipes mycelia.

• Archives of Pharmacal Research
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• v.27 no.6
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• pp.633-639
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• 2004

The expression of therapeutic transgenes in recombinant adenoviral vectors is a major cause of toxicity in dividing cancer cells as well as non dividing normal cells. To solve the problem of toxicity to normal cells, we have reported on a recombinant adenoviral vector system （AdLP-） in which the expression of the transgene is directed by the tumor-specific L-plastin promoter （LP） （Chung et al., 1999）. The object of this study was to generate a recombinant adenoviral vector system which would generate tumor cell specific expression of cytosine deaminase （CD） gene. We report the construction of a replication-incompetent adenoviral vector in which CD is driven by the L-plastin promoter （AdLPCD）. Infection of 293 cells by AdLPCD generated the functional CD protein as measured by HPLC analysis for the conversion of 5-Fluorocy-tosine （5-FC） to 5-Fluorouracil （5-FU）. HPLC analysis in conjunction with counting radioactivity for ［6-$^3$H］-5FC and ［6-$^3$H］-5FU demonstrated vector dose-dependent conversion of 5-FC to 5-FU in AdLPCD infected ovarian cancer cells. The results from present and previous studies（Peng et al., 2001； Akbulut et al., 2003） suggest that the use of the AdLPCD／5-FC system may be of value in the treatment of cancer including microscopic ovarian cancer in the peritoneal cavity.

• Biomedical Science Letters
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• v.9 no.2
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• pp.67-74
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• 2003

Viral and non-viral vectors have been used in the delivery of genetic materials into animal cells and tissues, with each approach having pros and cons. Non-viral vectors have many useful merits such as easy preparation, low immunity and size tolerance of a transgene when compared to those of viral vectors. Delivery specificity may be achieved by complex formation between receptor ligands and a non-viral vector. In the present study, non-viral vector systems are investigated in an effort to find a practical delivery means for gene therapy, Receptor-ligand interaction between transferrin-receptor and transferrin was utilized for efficient gene transfer into cancer cells. A plasmid vector, pcDNA3 (LacZ) was ligated with a small duplexed oligo fragment in which a Biotin- VN$^{TM}$ phosphoramidite was placed in the middle of the oligo. The plasmid vector labeled by biotin was then conjugated with biotin-labeled transferrin via streptavidin. This trimeric conjugates were delivered to a hepatoma cell line, HepG2. The delivery efficiency of the trimeric conjugate was 2-fold higher than that of cationic liposomes used for transfection of a plasmid vector. These results demonstrate that a plasmid vector can be efficiently transferred into cells by forming a trimeric complex of plasmid vector-linker-ligand.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2003.04a
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• pp.126-126
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• 2003

Sucrose-phosphate synthase (SPS) is a key regulatory enzyme in sucrose synthesis. To investigate the role of SPS in carbon partitioning, we produced transgenic rice plants overexpressing a cyanobacterial SPS from Synechocystis sp. PCC 6803. The gene was expressed under the control of the maize Ubil promoter in transgenic plants. Southern and Northern blot analyses confirmed the integration and the expression of the transgene in four transgenic rice lines. All of the four transgenic! lines analyzed showed abnormal vegetative and reproductive developments. Analysis of SPS activities and primary metabolites in the transgenic rice plants will be presented.

• Journal of Crop Science and Biotechnology
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• v.10 no.2
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• pp.73-78
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• 2007

The green revolution has significantly helped in increasing the food production. So far, various breeding methods have been exploited, besides them recombination DNA technology provides another approach for increasing the food production. By means of this technology the losses in food production incurred by various biotic and abiotic stresses can be effectively controlled. In most of the transgenic studies scientists have used antibiotic resistant genes as markers for easy selection of transformants but there are risks involved in use of GM foods. To make such foods safer and environment friendly we have discussed a novel strategy i.e. Cre-lox which involves site specific recombination. By means of Cre-lox the marker genes can be specifically removed once the selection of transformants is over. In addition, this strategy can be used to module the hybrid chromosomes, avoid gene silencing and incorporate single copy of a transgene for its higher expression.

• Korean Journal of Plant Tissue Culture
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• v.22 no.4
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• pp.201-205
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• 1995

The synthetic oligomers called nos right palindrome (RP) element and left palindrome (LP) element were inserted into nos.minimal promoter nos 5'-101 deletion mutant The activity of nos promoter was measured by studying the expression pattern of gene fusion between nos promoter and reporter genes such as chloramphenicol acetyltransferase and $\beta$-glucuconidase. Analysis of transgenic tobacco plane carrying transgene showed that the activity of nos minimal promoter activity was recovered by insertion of synthetic nos RP element. Nos RP element insertion of nos minimal promoter was induced by auxin, dithiothreitol, salicylic acid and methyl jasmonate.

• Plant Biotechnology Reports
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• v.2 no.1
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• pp.47-58
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• 2008

To select agronomically useful transgenic plants, a large number of transgenic events are initially produced, gene transfer confirmed, and advanced to obtain homozygous lines for testing in field trials. Direct in planta assays for identifying the transgene carriers in the segregating progeny are based on the activity of selectable marker gene and are easy, simple and inexpensive. For this purpose, expression of bar gene as measured by tolerance to damage by glufosinate ammonium, the active ingredient in the herbicide BASTA, was investigated. Dose damage curves were generated by leaf paint tests with BASTA on four genotypes of sorghum. Transgenic plants were characterized in terms of sensitivity to the concentration of glufosinate ammonium. In transgenics, symptoms of BASTA swab tests at different growth stages and PCR analysis for cry1B were carried out and correlated. Germination tests could not be employed for large scale evaluation of transgenic progeny because of mortality of tolerant seedlings after transplantation to soil. Based on the above findings, a simple, inexpensive, time-saving, two-step scheme for effective evaluation of transgenics and their progeny containing bar gene as selection marker using BASTA swab tests is described.