• Environmental Analysis Health and Toxicology
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• 제14권1_2호
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• pp.1-12
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• 1999

Recently, several new methods for the detection of genetic damages in vitro and in vivo based on molecular biological techniques were introduced according to the rapid progress in toxicology combined with cellular and molecular biology. Among these methods, mouse lymphoma thymidine kanase (tk) gene forward mutation assay, single cell gel electrophoresis (comet assay) and transgenic animal and cell line model as a target gene of lac I (Big Blue) and lac Z (Muta Mouse) gene mutation are newly introduced based on molecular toxicological approaches. The mouse lymphoma tk$\^$+/-/ gene assay (MOLY) using L5178Y tk$\^$+/-/ mouse lymphoma cell line is one of the mammalian forward mutation assays, and has many advantages and more sensitive than hprt assay. The target gene of MOLY is a heterozygous tk$\^$+/-/ gene located in 11 chromosome, so it is able to detect the wide range of genetic changes like point mutation, deletion, rearrangement, and mitotic recombination within tk gene or deletion of entire chromosome 11. The comet assay is a rapid, simple, visual and sensitive technique for measuring and analysing DNA breakages in mammalian cells, Also, transgenic animal and cell line models, which have exogenous DNA incorporated into their genome, carry recoverable shuttle vector containing reporter genes to assess endogenous effects or alteration in specific genes related to disease process, are powerful tools to study the mechanism of mutation in vivo and in vitro, respectively. Also in vivo acridine orange supravital staining micronucleus assay by using mouse peripheral reticulocytes was introduced as an alternative of bone marrow micronucleus assay. In this respect, there was an International workshop on genotoxicity procedure (IWGTP) supported by OECD and EMS (Environmental Mutagen Society) at Washington D. C. in March 25-26, 1999. The objective of IWGTP is to harmonize the testing procedures internationally, and to extend to finalization of OECD guideline, and to the agreement of new guidelines under the International Conference of Harmonization (ICH) for these methods mentioned above. Therefore, we introduce and review the principle, detailed procedure, and application of MOLY, comet assay, transgenic mutagenesis assay and supravital staining micronucleus assay.

• Toxicological Research
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• 제21권1호
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• pp.1-14
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• 2005

Transgenic mouse models have been introduced and accepted by regulatory bodies as an alternative to carcinogenicity assay models to predict and evaluate chemical carcinogens. The recent research outcomes in transgenic mouse models have made progressive advances in the understanding of chemical carcinogenesis and the evaluation of potential human carcinogens. However, these models still remain to be insufficient assay systems although the insufficiencies have been recognised and are being resolved. Based on up to date information from literature, this review article intends to understand currently accepted transgenic mouse models, issues arising from study design, interpretation of the study, results of validation project and their cancer prediction rate, and further perspectives of cancer assay models from the regulatory view point.

• Reproductive and Developmental Biology
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• 제31권4호
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• pp.253-260
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• 2007

To examine the differential protein expression pattern in the 11.5 day post-coitus (dpc) and 18.5 dpc placenta of mouse, we have used the global proteomics approach by 2-D gel electrophoresis (2-DE) and MALDI-TOF-MS. The differential protein patterns of 3 placentae at the 11.5 dpc and 18.5 dpc from nature mating mice were analyzed. Proteins within isoelectric point range of $3.0{\sim}10.0$, separately were analyzed in 2DE with 3 replications of each sample. A total of approximately 1,600 spots were detected in placental 2-D gel stained with Coomassie-blue. In the comparison of 11.5 dpc and 18.5 dpc placentae, a total of 108 spots were identified as differentially expressed proteins, of which 51 spots were up-regulated proteins such as alpha-fetoprotein, mKIAA0635 protein and transferrin, annexin A5, while 48 spots were down-regulated proteins such as Pre-B-cell colony-enhancing factor l(PBEF), aldolase 1, A isoform, while 4 spots were 11.5 dpc specific proteins such as chaperonin and Acidic ribosomal phosphoprotein P0, while 3 spots were 18.5 dpc specific proteins such as aldo-keto reductase family 1, member B7 and CAST1/ERC2 splicing variant-1. Most identified proteins in this analysis appeared to be related with catabolism, cell growth, metabolism and regulation. Our results revealed composite profiles of key proteins involved in mouse placenta during pregnancy.

• IMMUNE NETWORK
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• 제14권4호
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• pp.219-225
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• 2014

We examined the immunogenicity of H-2 class I-restricted and HLA-A2-restricted epitopes through peptide immunization of HLA-A2-transgenic mice that also express mouse H-2 class I molecules. All four of the tested epitopes restricted by H-2 class I robustly elicited T-cell responses, but four of seven epitopes restricted by HLA-A2 did not induce T-cell responses, showing that HLA-A2-restricted peptide epitopes tend to be poorly immunogenic in HLA-A2-transgenic mice. This finding was confirmed in HLA-A2-transgenic mice infected with a recombinant vaccinia virus expressing hepatitis C virus proteins. We examined the precursor frequency of epitope-specific naïve $CD8^+$ T cells in HLA-A2-transgenic and conventional C57BL/6 mice and found that the poor immunogenicity of HLA-A2-restricted peptide epitopes is related to the paucity of naïve $CD8^+$ T-cell precursors in HLA-A2-transgenic mice. These results provide direction for the improvement of mouse models to study epitope repertoires and the immunodominance of human T-cell responses.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2000년도 국제심포지움
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• pp.7-8
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• 2000

Transgenic animals are very useful for scientific, pharmaceutical, and agricultural purposes. In livestock, transgenic technology has been used forthe genetic alteration of farm animals, the production of human proteins inlarge quantities in the milk of transgenic farm animals, and the generation of animals with organs suitable for xenotransplantation. To date, the transfer of foreign genes into farm animals has been performed mainly by microinjection of DNA into the pronuclei of fertilized eggs. However, the overall success rate of transgenic animals in livestock so far has been disappointingly low, eg., the efficiency is 0∼5% in swine, and less than 1% in sheep and cattle, compared with the rate in mice where 5% microinjected develop into transgenic animals. Recently, McGreath et al. (2000) have succeeded in producing the gene targeted sheep by the use of nuclear transfer from cultured somatic cells transfected with a foreign gene in vitro. However, we may need plenty of time until currently employ this method for gene transfer to farm animals. We have been studying to exploit the method for improving production efficiencies of transgenic animals with emphasis of its application to farm animals. The present paper describes three approaches that we have made in our laboratory to improve production efficiencies of transgenic animals, based on the DNA microinjection method. 1. Co-injection of restriction enzyme with foreign DNA into the pronucleus for elevating production efficiencies of transgenic animals. 2. Efficient selection of transgenic mouse embryos using EGFP as a marker gene. 3. Phenotypes of tansgenic mice expressing WAP/hGH-CAG/EGFP fusion gene produced by selecting transgenic embryos. 4. Efficient site-specific integration of the transgene targeting an endogenous lox like site in early mouse embryos.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.99-99
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• 2002

The hormone resistin is associated with typeII diabetes mellitus in rodent model. Resistin impairs glucose tolerance and insulin action. A new class of anti-diabetic drugs were called thiazolidinediones (TZDs) downregulates a resistin which is induced during adipocyte differentiation. But the connection between increased adiposity and resistin remains unknown. The objectives of this study was to clone a mouse resistin cDNA and to generate transgenic mice overexpressing mouse resistin gene. The 555 bp of mouse resistin was amplified from mob cDNAS by polymerase chain reaction (PCR) and cloned into pCR$\^$(R)/ 2.1 TOPO T-vector. Mouse resistin mRNA on the basis of Genbank sequence (acession no. AF323080). Then, the PCR product was cloned into pTargeT$\^$TM/ mammalian expression vector that has pCMV promoter and chimeric intron. Restriction enzyme analysis with BamH I and Not I was carried out to determine an orientation of the insert in the vector. The pCMV-mus/resistin gene was prepared from previous recombinant pTargeT$\^$TM/-mus/resistin by digestion of Bgl II, and has used for microinjection into pronuclei of one cell embryos. The microinjected embryos were transfered to pseudopregnant foster-mother. Mouse resistin expression was detected in transgenic F1 mice by Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR). Resistin gene expression mouse has heavier body weight which was measured higher level of plasma glucose than that of normal mouse. And in diet-induced experiments, the abdominal fat pads were isolated from each 24h starvation and re-feeding after fasting group mice that were assessed by RT-PCR analysis. In fasting group mice, resistin expression was higher than that of re-feeding group mice. This result suggests that the resistin gene overexpressing mice may be became to obesity and be useful as an animal disease model to be diabetes mellitus caused by insulin resistance of resistin.

• BMB Reports
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• 제37권1호
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• pp.122-132
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• 2004

The rapid development and characterization of the mouse genome sequence, coupled with comparative sequence analysis of human, has been paralleled by a reinforced enthusiasm for mouse functional genomics. The way to uncover the in vivo function of genes is to analyze the phenotypes of the mutant animals. From this standpoint, the mouse is a suitable and valuable model organism in the studies of functional genomics. Therefore, there have been enormous efforts to enrich the list of the mutant mice. Such a trend emphasizes the random mutagenesis, including ENU mutagenesis and gene-trap mutagenesis, to obtain a large stock of mutant mice. However, since various mutant alleles are needed to precisely characterize the role of a gene in vivo, mutations should be designed. The simplicity and utility of transgenic technology can satisfy this demand. The combination of RNA interference with transgenic technology will provide more opportunities for researchers. Nevertheless, gene targeting can solely define the in vivo function of a gene without a doubt. Thus, transgenesis and gene targeting will be the major strategies in the field of functional genomics.

• Reproductive and Developmental Biology
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• 제34권2호
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• pp.63-71
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• 2010

The spontaneous mutant circling mouse (cir/cir) shows a circling behavior and hearing loss. We produced transgenic mice overexpressing transmembrane inner ear (tmie) gene, the causative gene, for the phenotypic rescue of the circling mouse. Through the continuous breeding with circling mice, the cir/cir homozygous mice carrying the transgene (cir/cir-tg) were produced. The rescued cir/cir-tg mice were able to swim in the water with proper orientation and did not show any circling behavior like wild type mice. Western blot and immunohistochemical analysis exhibited that the transgenic tmie was expressed in the inner ear. Inner and outer hair cells were recovered in the cochlea and spiral ganglion neurons were also recovered in the rescued mice. Auditory brainstem response (ABR) test demonstrated that the cir/cir-tg mice are able to respond to sound. This study demonstrates that tmie transgene can recover the hearing impairment and abnormal behavior in the circling mouse.

• 식물조직배양학회지
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• 제22권4호
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• pp.195-200
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• 1995

동물유전자인 mouse adenosine deaminase (ADA)유전자가 안정적으로 연초 형질전환체에서 발현되었다. ADA cDNA 을 연초에서 강력히 발현시키기 위해서 35S/35S/AMV promoter을 부착시켰으며 식물세포에 도입을 위해서 binary vector인 pRD400을 이용하였다. 연초의 형질전환은 Tri- parental mating에 의해서 도입된 ADA 유전자 함유 binary vector와 disarmed Ti-plasmid을 함유하고 있는 Agrobacterium tmefacience MP9O을 사용하였다. 동시배양은 연초의 잎 disc을 이용해서 kanamycin이 첨가된 배지로부터 직접 shoots을 선발하여 형질전환체로 유도하였다. 형질전환 체에 ADA 유전자의 삽입여부는 PCR을 이용하였으며, 실험결과 ADA 유전자를 확인할 수 있었다. 또한 도입된 mouse ADA 유전자로부터 mRNA 및 단백질 합성여부를 각각 northern blot 및 immunoblot 분석한 결과 형질전환체 에서는 공히 확인되었다.

• Reproductive and Developmental Biology
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• 제33권4호
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• pp.237-242
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• 2009

Pregnancy is a unique event in which a fetus develops in the uterus despite being genetically and immunologically different from the mother, and the underlying mechanisms remain poorly understood. To analyze the differential gene expression profiles in nonpregnant and 7 days post coitus (dpc) pregnant uterus of mice, we performed a global proteomic study by 2-D gel electrophoresis (2-DE) and MALDI-TOF-MS. The uterine proteins were separated using 2-DE, Approximately 1,000 spots were detected on staining with Coomassie brilliant blue. An image analysis using Melanie III (Swiss Institute for Bioinformatics) was performed to detect variations in protein spots between pregnant and nonpregnant uterus. Twenty-one spots were identified as differentially expressed proteins, of which 10 were up-regulated proteins such as alpha-fetoprotein, chloride intracellular channel 1, transgelin, heat-shock protein beta-1, and carbonic anhydrase II, while 11 were down-regulated proteins such as X-box binding protein, glutathione S-transferase omega 1, olfactory receptor Olfr204, and metalloproteinase-disintegrin domain containing protein TECADAM. Most of the identified proteins appeared to be related with catabolism, cell growth, metabolism, regulation, cell protection, protein repair, or protection. Our results uncovered key proteins of mouse uterus involved in pregnancy.