• Korean Journal of Animal Reproduction
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• v.17 no.3
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• pp.263-267
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• 1993

Transgenic animals have become an important tool in the basic and applied sectors of genetic and biomedical sciences. In particular transgenes provide clear-cut markers in the spatial and temporal analysis of developing embryos for the understanding of developmental mechanisms. For the long-term use of plasmid vector in a particular purpose it would be necessary to develop one's own vector system which can be properly expressed in eukaryotic system. Plasmids were constructed from ori region of pUC19 and early region of SV40 through various steps. LacZ gene coding for $\beta$-galactosides was fused to early gene of SV40 in translational in-frame. Poly(A) tailing site of SV40 was inserted at the 3' lacZ so that initiation, elongation and terminatin be controlled by SV40 transcription (pSS4). Biological function of the constructed pSS4 was demonstrated via microinjection of the plasmid into fertilized loach eggs and subsequent detection of $\beta$-galactosidase in developing embryos. The result indicate that the newly constructed pSS4 is functional in a eukaryotic system in vivo. Thus pSS4 may be used as an efficient tool for the study of embryogenesis and a basic carrier for various genes for animal transgenesis.

• Korean Journal of Animal Reproduction
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• v.22 no.2
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• pp.145-152
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• 1998

Many trials have been made to produce transgenic animals using sperm cells as a vector transferring foreign DNA into eggs, but reliable results are yet to be obtained (Brinster et al., 1989; Lavitrano et al., 1989; Bachiller et al., 1991; Sato et al., 1994). Recently, one of author(SO) demonstrated that mouse blastocysts derived from eggs fertilized by spermatozoa of male mice single injected with liposome-DNA complexes within the testis expressed thegene (Ogawa et al., 1995.) Here we report that a single injection of liposome-encapsulated DNAs into the testis of either male rats or mice resulted in successfully gene transfer to the postpartum progeny. The expression of mRNA derived from transgenes was also demonstrated in transgenic animals thus obtained. Further, the transmission of the exogenous gene to the descedants was confirmed in one line of transgenic rat up to F4 generation, indicating that the gene was stably incorporated into the germ line. Thus, direct single injection of foreign DNA into the testis provides a novel and convenient means to generate transgenic animals.

• 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.

• Molecules and Cells
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• v.39 no.4
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• pp.358-366
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• 2016

The digestive system is gaining interest as a major regulator of various functions including immune defense, nutrient accumulation, and regulation of feeding behavior, aside from its conventional function as a digestive organ. The Drosophila midgut epithelium is completely renewed every 1-2 weeks due to differentiation of pluripotent intestinal stem cells in the midgut. Intestinal stem cells constantly divide and differentiate into enterocytes that secrete digestive enzymes and absorb nutrients, or enteroendocrine cells that secrete regulatory peptides. Regulatory peptides have important roles in development and metabolism, but study has mainly focused on expression and functions in the nervous system, and not much is known about the roles in endocrine functions of enteroendocrine cells. We systemically examined the expression of 45 regulatory peptide genes in the Drosophila midgut, and verified that at least 10 genes are expressed in the midgut enteroendocrine cells through RT-PCR, in situ hybridization, antisera, and 25 regulatory peptide-GAL transgenes. The Drosophila midgut is highly compartmentalized, and individual peptides in enteroendocrine cells were observed to express in specific regions of the midgut. We also confirmed that some peptides expressed in the same region of the midgut are expressed in mutually exclusive enteroendocrine cells. These results indicate that the midgut enteroendocrine cells are functionally differentiated into different subgroups. Through this study, we have established a basis to study regulatory peptide functions in enteroendocrine cells as well as the complex organization of enteroendocrine cells in the Drosophila midgut.

• Plant Biotechnology Reports
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• v.2 no.1
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• pp.13-20
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• 2008

Potato (Solanum tuberosum L.), one of the most important food crops, is susceptible to a number of devastating fungal pathogens in addition to bacterial and other pathogens. Producing disease-resistant cultivars has been an effective and useful strategy to combat the attack of pathogens. Potato was transformed with Agrobacterium tumefaciens strain EHA101 harboring chitinase, (ChiC) isolated from Streptomyces griseus strain HUT 6037 and bialaphos resistance (bar) genes in a binary plasmid vector, pEKH1. Polymerase chain reaction (PCR) analysis revealed that the ChiC and bar genes are integrated into the genome of transgenic plants. Different insertion sites of the transgenes (one to six sites for ChiC and three to seven for bar) were indicated by Southern blot analysis of genomic DNA from the transgenic plants. Expression of the ChiC gene at the messenger RNA (mRNA) level was confirmed by Northern blot analysis and that of the bar gene by herbicide resistance assay. The results obviously confirmed that the ChiC and bar genes are successfully integrated and expressed into the genome, resulting in the production of bialaphos-resistant transgenic plants. Disease-resistance assay of the in vitro and greenhouse-grown transgenic plants demonstrated enhanced resistance against the fungal pathogen Alternaria solani (causal agent of early blight).

• Korean Journal of Breeding Science
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• v.43 no.4
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• pp.252-259
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• 2011

Conventional staining and fluorescence in situ hybridization (FISH) karyotypes of the non-genetically modified (GM) parental rice line, 'Nakdong' (Oryza sativa L. japonica), and its four GM rice lines, LS28 (event LS30-32-20-1), Cry1Ac1 (event C7-1-9-1), and LS28 ${\times}$ Cry1Ac1 (events L/C1-1-3-1 and L/C1-3-1-1) were analyzed using 5S and 45S rDNAs as probes. Both parental and transgenic lines were diploids (2n=24) with one satellite chromosome pair. The lengths of the prometaphase chromosomes ranged from 1.50 to $6.30{\mu}m$. Four submetacentric and eight metacentric pairs comprised the karyotype of 'Nakdong' and its four GM lines. One pair of 5S rDNA signals was detected near the centromeric region of chromosome g in both the parental and transgenic lines. The 45S rDNA signals were detected on the secondary constrictions of the satellite chromosome pair in both the parental and transgenic lines. There was no significant difference in chromosome size, length, and composition between 'Nakdong' and its four GM lines. This research was conducted as a preliminary study for chromosomal detection of transgenes in GM rice lines and would be useful for their breeding programs.

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.6
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• pp.856-860
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• 2000

Cloning technology continues to capture widespread attention by the international news media and biomedical and agricultural industries. The future uses of this technology could potentially contribute to major advances in biomedical and agricultural sciences. Cloned transgenic dairy cattle possessing milk promoters directing transgenes will produce pharmaceutical proteins in their milk faster, more efficiently and less expensively than transgenic cattle created using microinjection techniques. Additionally, cloned transgenic fetuses and animals may become a source of cells, tissue and organs for xenotransplantation. Lastly, but maybe most importantly, enhanced production traits and disease resistance may be realized in animal agriculture by utilizing these new technologies. The recent advances in the cattle cloning technology are important but there are still major obstacles preventing widespread commercial use of this technology. The type of donor nucleus, recipient cytoplasm, and cloning procedures used will impact the potential number of clones produced and the uses of the technology. In addition, the new advances in cloning methodology have not improved the relatively low pregnancy rates or reduced the incidence of health problems observed in cloned offspring. These problems may require novel techniques to decipher their cause and new methods of preventing and/or diagnosing them in the preimplantation embryo. The commercial potential is enormous for cloning technology; however, little has been done to improve the efficiencies of the procedure. Improving procedural efficiencies is a critical developmental milestone especially for potential uses of cloning technology in animal agriculture.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.6
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• pp.886-892
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• 2017

Objective: Transgenic technology is widely used for industrial applications and basic research. Systems that allow for genetic modification play a crucial role in biotechnology for a number of purposes, including the functional analysis of specific genes and the production of exogenous proteins. In this study, we examined and verified the cumate-inducible transgene expression system in chicken DF1 and quail QM7 cells, as well as loxP element-mediated transgene recombination using Cre recombinase in DF1 cells. Methods: After stable transfer of the transgene with piggyBac transposon and transposase, transgene expression was induced by an appropriate concentration of cumate. Additionally, we showed that the transgene can be replaced with additional transgenes by co-transfection with the Cre recombinase expression vector. Results: In the cumate-GFP DF1 and QM7 cells, green fluorescent protein (GFP) expression was repressed in the off state in the absence of cumate, and the GFP transgene expression was successfully induced in the presence of cumate. In the cumate-MyoD DF1 cells, MyoD transgene expression was induced by cumate, and the genes controlled by MyoD were upregulated according to the number of days in culture. Additionally, for the translocation experiments, a stable enhanced green fluorescent protein (eGFP)-expressing DF1 cell line transfected with the loxP66-eGFP-loxP71 vector was established, and DsRed-positive and eGFP-negative cells were observed after 14 days of co-transfection with the DsRed transgene and Cre recombinase indicating that the eGFP transgene was excised, and the DsRed transgene was replaced by Cre recombination. Conclusion: Transgene induction or replacement cassette systems in avian cells can be applied in functional genomics studies of specific genes and adapted further for efficient generation of transgenic poultry to modulate target gene expression.

• Development and Reproduction
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• v.8 no.1
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• pp.19-25
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• 2004

Pax6, a member of the highly conserved homeobox gene family, is known to be expressed in spatially and temporally restricted pattern during embryogenesis. To examine the spatial expression pattern of Pax6 in malaria vector mosquito Anopheles stephemi, in different molecular environment, the germ line transformation technique using piggyBac transposon combined with the use of Pax6 specific 3xp3-EGFP marker was utilized. Four transgenic lines with a transformation rate of 6.7% were established. Transgenes were stably expressed in subsequent several generations. The transgenic lines showed 3 different expression pattern with spatial specificity, possibly due to enhancing and/or silencing position effects. In two transgenic lines, noble expression pattern of Pax6 was observed in the region that has not been previously reported in any animal species. The results show that the tranposon piggyBac mediated germ line transformation system can be used as an efficient tool for the generation of diverse spatially restricted reporter gene expression.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.43-43
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• 2003

This study was conducted to produce transgenic pig harboring human tissue plasminogene activator (tPA) gene. Two different tPA genes containing bovine $\beta$-casein promoter and mouse uroplakin promoter were prepared for microinjection and confirmed the expression level of tPA protein from the CHO (Chinese hamster ovary) cell lines by gene transfection. Concentration of tPA expression from the six cell lines (all of CHO cells) were average 212.4 ng/ml. Reconstructed DNA to used the CHO cell were microinjected into the pronuclei of in vivo embryos The total of 2,307 zygotes were collected from 95 donors and 1,851 embryos were in 1-cell stage which were visualized the pronuclei for DNA microinjection. The concentration of linear DNA was 2.0 ng per microliter and injected into zygotes with two pronuclei on an inverted Nikon microscope equipped with narishige micromanipulator and modulation contrast optics. The 541 embryos injected with bovine $\beta$-casein promoter-tPA were transferred to 22 recipients. The 1,154 embryos injected with mouse uroplakin promoter-tPA were transferred to 51 recipients. Sixty nine offspring from 9 delivered sows were produced. We analysed the transgenes with PCR methods from 69 offsprings, but could not detect the PCR product from piglet tails DNA.