• Asian-Australasian Journal of Animal Sciences
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• v.21 no.5
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• pp.745-753
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• 2008

Gene-manipulated mice were discovered for the first time about a quarter century ago. Since then, numerous sophisticated technologies have been developed and applied to answer key questions about the fundamental roles of the genes of interest. Functional genomics can be characterized into gain-of-function and loss-of-function, which are called transgenic and knock-out studies, respectively. To make transgenic mice, the most widely used technique is the microinjection of transgene-containing vectors into the embryonic pronucleus. However, there are critical drawbacks: namely position effects, integration of unknown copies of a foreign gene, and instability of the foreign DNA within the host genome. To overcome these problems, the ROSA26 locus was used for the knock-in site of a transgene. Usage of this locus is discussed for the gain of function study as well as for several brilliant approaches such as conditional/inducible transgenic system, reproducible/inducible knockdown system, specific cell ablation by Cre-mediated expression of DTA, Cre-ERTM mice as a useful tool for temporal gene regulation, MORE mice as a germ line delete and site specific recombinase system. Techniques to make null mutant mice include complicated steps: vector design and construction, colony selection of embryonic stem (ES) cells, production of chimera mice, confirmation of germ line transmission, and so forth. It is tedious and labor intensive work and difficult to approach. Thus, it is not readily accessible by most researchers. In order to overcome such limitations, technical breakthroughs such as reporter knock-in and gene knock-out system, production of homozygous mutant ES cells from a single targeting vector, and production of mutant mice from tetraploid embryos are developed. With these upcoming progresses, it is important to consider how we could develop these systems further and expand to other animal models such as pigs and monkeys that have more physiological similarities to humans.

• Animal cells and systems
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• v.1 no.4
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• pp.657-663
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• 1997

The CD4 and CDS coreceptors, in conjunction with the T cell receptor (TCR) , make important contributions to the differentiation of thymocytes. They have been shown to be involved in the clonal deletion and positive selection processes during T cell development in thymus. To further analyze the role of CD4 and CDS proteins during T cell differentiation, we have generated transgenic mice constitutively expressing high levels of a native CD4 and a CD4{CDSa hybrid protein. The hybrid protein is composed of CD4 extracellular domain linked to the CD8a transmembrane region and cytoplasmic tail. The transgenes were driven by human beta-actin promoter, and therefore, they were expressed in all tissues examined including thymus, spleen, and lymph nodes. The resulting CD4 and CD4{CD8${\alpha}$transgenic mice were found to express the CD4 and CD4{CD8${\alpha}$ respectively, in developing thymocytes and peripheral T cells. The expression levels of transgenic proteins were 5-10 times higher than that of endogenous CD4 in thymus. However, total surface CD4 expression (CD4 or CD4{CD8${\alpha}$ transgenic protein plus endogenous CD4) of the transgenic mice were main. tained at similar levels compared to control littermates. Surface CD4 expression on CDS T cells, however, was significantly lower than that on cells expressing endogenous CD4. These results suggest that a total avidity between developing thymocytes and thymic stromal cells is impor. tant for differentiation of thymocytes.

• Parasites, Hosts and Diseases
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• v.37 no.2
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• pp.93-99
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• 1999

Eosinohil and IgE responses of interleukin IL(-5 transgenic and normal C3H/HeN mice were studied after experimental infection with Nippostrongylus brasiliensis 9Nb). Intestinal worms were recovered at day 5 post-infection (PI), and numbers of total white blood cells (WBC) and eosinophils, and total serum IgE and anti-hapten (dinitrophenyl)(DNP) specific IgE titers, were measured at days 0,14 and PI. IL-5 mice appeared resistant to Nb infection showing a significantly ower worm recovery rate than normal mice (P<0.05). Total WBC and eosinophil counts (/mm3) were significantly increased in Nb infected normal mice (p<0.05), but unchanged (total WBC) or decreased (eosinophils) in IL-5 mice at day 21 PI. The total serum IgE level remarkably increased in normal mice, but only a little in IL-5 mice at days 14 and 21 PI. Priming with DNP brought about more remarkable increases of the total and anti-DNP specific IgE in normal mice than in IL-5 mice. The results show that IL-5 mice are resistant to Nb infection, and that eosinophil and IgE responses in these mice are not augmented by N infection.

• Journal of Microbiology and Biotechnology
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• v.23 no.3
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• pp.405-413
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• 2013

Monocyte chemotactic protein-3 (MCP-3), a chemokine that is in a superfamily of structurally related small chemotactic cytokines involved in leukocyte trafficking, is regarded as a key factor in atherogenesis. In this study, we examined the changes in atherogenic parameters including hepatic lipid accumulation and oxidative balance in MCP- 3-overexpressing transgenic mice (MCP-3 mice) under atherogenic conditions. To induce an extreme atherogenic condition, mice were fed a high-fat, high-cholesterol (HFHC) diet for 12 weeks. The body weight and food intake were not changed by MCP-3 overexpression in the aorta. On a HFHC diet, the MCP-3 mice had higher plasma levels of total cholesterol and a higher atherogenic index compared with wild-type mice, although there were no differences in the plasma HDL-cholesterol and triglyceride levels. Furthermore, an increase in lipid accumulation was observed in the aortas as well as the livers of the HFHC diet-fed MCP-3 mice compared with wild-type mice. The activities of antioxidant enzymes increased in the livers of the HFHC diet-fed MCP-3 mice, whereas supplementation with antioxidants, naringin and hesperidin, reversed the activities of the hepatic antioxidant enzymes in HFHC diet-fed MCP-3 mice, indicating that there might be more oxidative damage to the tissues in the HFHC diet-fed MCP-3 mice leading to progression towards atherosclerosis and hepatic steatosis. Microarray analyses of the aorta revealed atherosclerosis-, PPARs-, lipoprotein receptor, and apolipoprotein-related genes that were affected by the HFHC diet in MCP-3 mice. These findings suggest that aortic MCP-3 overexpression may contribute to the development of atherosclerosis and hepatic steatosis under atherogenic conditions.

• Journal of Life Science
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• v.16 no.4
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• pp.566-570
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• 2006

Pronuclear microinjection (PMI) is a primary method for producing transgenic mice and offers a powerful tool for investigating gene function in vivo. The method has several reported advantages and disadvantages. Here, we report another potential shortcoming. The survival rate of fertilized one cell-stage embryos was significantly reduced after PMI procedure (65.4% (1202/1838)). In addition, the proportion of embryos developing to full-term was also significantly lower than that of embryos not undergoing PMI (26.5% (319/1202) vs 41.9% (57/136)). Moreover, 3 out of 21 (14.3%) founder control mice which were non-transgene-carrying littermates of transgenic founders showed histopathological changes in their liver, which was comparable to that in of transgenic lineages (4 out of 27 (14.8%)). In conclusion, the mechanical damages in chromosomes occurring during PMI procedure may be a potential factor influencing phenotypes of transgenic mice.

• Korean Journal of Animal Reproduction
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• v.27 no.1
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• pp.9-14
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• 2003

The pBT-L transgenic mice carrying human TPO gene in conjunction with bovine $\beta$-casein promoter express human TPO in milk during lactation. In this study, stability of germ line transmission and expression of pBT-L transgene integrated into host chromosome were monitored up to generation F10 of transgenic pBT-L/15 line. When male mouse of generation F8 was crossbred with normal females, approximately half of offsprings (51.3$\pm$18.98%) were identified as transgenic mice. Generation F9 and F 10 mice also showed similar transmission rates (43.8$\pm$18.98% and 71.4$\pm$26.98%, respectively), implying that pBT-L transgene can be transmitted stably up to long term generation in the transgenic mice. Expression levels of human TPO from milk of generation F9 and F10 mice were 1.1$\pm$0.33 mg/ml and 1.1 $\pm$0.45 mg/ml, respectively, which are similar to expression level of generation F2 mice. In conclusion, our results suggest that transgenic animals once established will continuously pass their transgenes to the progeny through the breeding program with the same productivity of human protein in their milk.

• Reproductive and Developmental Biology
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• v.35 no.4
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• pp.415-420
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• 2011

The experimental manipulation of protooncogenes and their gene products is a valuable research tool for the study of human neoplasia. In this study, the recently identified human cervical cancer protooncogene (HccR-2) was expressed in transgenic mice under the control of the tetracycline regulatory system. Mice expressing the HccR-2 transgene showed an altered myeloid development characterized by an increased percentage of mature and band-form neutrophils in the peripheral blood, liver and spleen. This phenotype is similar to human chronic neutrophilic leukemia (CNL) in many ways, which is a rare chronic myeloproliferative disorder (CMD) that presents as a sustained leukocytosis of mature neutrophils with a few or no circulating immature granulocytes, an absence of peripheral blood monocytosis, basophilia, or eosinophilia, and an infiltration of neutrophils into the liver, spleen and kidney. Thus, the HccR-2 transgenic mouse model is imperative not only for investigating the biological properties of the HccR-2 protooncogene in vivo, but also for analyzing the mechanisms involved in the progression of CNL.

• Korean Journal of Veterinary Research
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• v.53 no.4
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• pp.217-224
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• 2013

Transgenic plants have been tested as an alternative host for the production and delivery of experimental oral vaccines. Here, we developed transgenic potatoes that express the major antigenic sites A and D of the glycoprotein S from transmissible gastroenteritis coronavirus (TGEV-$S_{0.7}$) under three expression vector systems. The DNA integration and mRNA expression level of the TGEV-$S_{0.7}$ gene were confirmed in transgenic plants by PCR and northern blot analysis. Antigen protein expression in transgenic potato was determined by western blot analysis. Enzyme-linked immunosorbent assay results revealed that based on a dilution series of Escherichia coli-derived antigen, the transgenic line P-2 had TGEV-$S_{0.7}$ protein at levels that were 0.015% of total soluble proteins. We then examined the immunogenicity of potato-derived TGEV-$S_{0.7}$ antigen in mice. Compared with the wild-type potato treated group and synthetic antigen treated group, mice treated with the potato-derived antigen showed significantly higher levels of immunoglobulin (Ig) G and IgA responses.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.121-121
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• 2003

The standard protocol for the production of transgenic mouse from ES-injected embryo has to process via chimera producing and several times breeding steps, In contrast, tetraploid-ES cell complementation method allows the immediate generation of targeted murine mutants from genetically modified ES cell clones. The advantage of this advanced technique is a simple and efficient without chimeric intermediates. Recently, this method has been significantly improved through the discovery that ES cells derived from hybrid strains support the development of viable ES mice more efficiently than inbred ES cells do. Therefore, the objective of this study was to generate transgenic mice overexpressing human resistin gene by using tetrapioid-ES cell complementation method. Human resistin gene was amplified from human fetal liver cDNA library by PCR and cloned into pCR 2.1 TOPO T-vector and constructed in pCMV-Tag4C vector. Human resistin mammalian expression plasmid was transfected into D3-GL ES cells by lipofectamine 2000, and then after 8~10 days of transfection, the human resistin-expressing cells were selected with G418. In order to produce tetraploid embryos, blastomeres of diploid embryos at the two-cell stage were fused with two times of electric pulse using 60 V 30 $\mu$sec. (fusion rate : 93.5%) and cultured upto the blastocyst stage (development rate : 94.6%). The 15~20 previously G418-selected ES cells were injected into tetraploid blastocysts, and then transferred into the uterus of E2.5d pseudopregnant recipient mice. To investigate the gestation progress, two El9.5d fetus were recovered by Casarean section and one fetus was confirmed to contain human resistin gene by genomic DNA-PCR. Therefore, this finding demonstrates that tetraploid-ES mouse technology can be considered as a useful tool to produce transgenic mouse for the rapid analysis of gene function in vivo.

• BMB Reports
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• v.44 no.8
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• pp.535-540
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• 2011

Reprogramming errors, which appear frequently in cloned animals, are reflected by aberrant gene expression. We previously reported the aberrant expression of TIMP-2 and PBEF in cloned placenta and differential expression of PBEF genes during pregnancy. To examine the epigenetic modifications that regulate dynamic gene expression in developing placentae, we herein analyzed the mRNA and protein expression levels of PBEF and TIMP-2 in the placentae of normal mice during pregnancy and then examined potential correlations with epigenetic modifications. DNA methylation pattern analysis revealed no difference, but ChIP assays using antibodies against H3-K9/K14 and H4-K5 histone acetylation revealed that the H3-K9/K14 acetylation levels, but not the H4-K5 acetylation levels, of the TIMP-2 and PBEF loci were significantly correlated with their gene expression levels during placentation in normal mice. These results suggest that epigenetic changes may regulate gene expression level in the developing placentae of normal mice and that inappropriate epigenetic reprogramming might be one cause of the abnormal placentae seen in cloned animals.