• Asian-Australasian Journal of Animal Sciences
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• 제21권5호
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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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• 제1권4호
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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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• 제37권2호
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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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• 제23권3호
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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.

• 생명과학회지
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• 제16권4호
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• pp.566-570
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• 2006

현재, 유전자의 in vivo 기능을 연구하기 위해 가장 많이 이용되고 있는 transgenic mice를 생산하기 위한 기본적으로 이용되고 있는 방법이 one cell-stage embryo에 pronuclear microinjection (PMI)이다. 그러나, 이 PMI 후에 one cell-stage embryo들의 생존율은 현저히 감소 (65.4%)할 뿐만 아니라 PMI 후의 embryo의 출생률(26.4%)이 PMI 처리를 하지 않은 것 (41.9%) 보다 현저히 낮다. 더욱이, PMI 방법에 의해 태어난 transgenic founder들의 간 조직에 병리학적 변화가 14.8% 정도에 대해서 같은 한배의 새끼 non-transgenic founder들의 경우도 간 조직에 병리학적 변화가 14.3%로 나타났다. 결론적으로, 이 PMI 방법에 의한 염색체에 물리적 손상은 형질전환 마우스의 생산 및 표현형에 영향을 미치는 잠재적 요소로 생각된다.

• 한국가축번식학회지
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• 제27권1호
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• pp.9-14
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• 2003

형질전환 동물의 유선에서 특이적으로 인간 TPO가 발현되도록 고안된 pBT-L 외래유전자가 삽입되어 한 계통으로 확립된 형질전환 생쥐에서 이 유전자가 장기 세대까지 안정적으로 전이되고, 또한 발현 수준도 지속적으로 유지되는지를 조사 하였다 이를 위해 제 5세대의 수컷 pBT-L 형질전환 생쥐를 실험에 공시하였고, 제 10세대까지의 전이율과 인간 TPO의 발현수준을 분석하였다. 제 8세대 생쥐의 계대 번식에 의한 자손 중 51.3$\pm$18.98%가 형질전환 생쥐로 판명되었다. 또한 제9세대에서 외래 유전자의 전이율은 43.8$\pm$18.98%이었고, 제 10세대에서는 71.4$\pm$26.98%의 전이율을 나타내었다. 이러한 결과로 pBT-L 형질전환 생쥐는 그 외래유전자의 유전적 손상이 없이 장기 세대까지 안정적으로 전이되는 것으로 판단된다. 제 9세대외 10세대의 3마리의 암컷 형질전환 생쥐로 부터 유즙내 인간 TPO의 발현 수준을 분석하였을때, 그 농도는 모두 평균 1.1 mg/ml의 수준에서 측정되었다. 이러한 수준은 제 2세대의 것과 유사한 결과로 pBT-L 형질전환 생쥐에서 인간 TPO 유전자의 발현은 최소한 10세대까지 지속적으로 유지된다는 것을 알 수 있었고, 더 긴 장기 세대까지도 발현 수준이 유지될 것으로 추정된다. 이러한 연구결과는 계통으로 확립된 형질전환동물에 부여된 새로운 유전형질은 장기 세대까지 유전될 수 있음을 보여주는 것이다.

• Reproductive and Developmental Biology
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• 제35권4호
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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.

• 대한수의학회지
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• 제53권4호
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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.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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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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• 제44권8호
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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.