• Development and Reproduction
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• v.14 no.4
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• pp.215-223
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• 2010

The construction of transgenic mouse using embryonic stem (ES) cells has been crucial in the functional studies of gene on mouse genome. Gene knockout mice have been powerful for elucidating the function of genes as well as a research model for human diseases. Gene targeting and gene trapping mathods have been the representative technologies for making the knockout mice by using ES cells. Since the gene targeting and the gene trapping methods were independently developed about 20 years ago, it's efficiency and productivity has been improved with a advance of molecular biology. Conventional gene targeting method has been changes to high throughput conditional gene targeting. The combination of the advantage of gene targeting and gene tapping elements allows to extend a spectrum of gene trapping and to improve the efficiency of gene targeting. These advance should be able to produce the mutant with various phenotype to target a certain gene, and in postgenome era they have served as crucial research tools in understanding the functional study of whole genome in mouse.

• BMB Reports
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• v.42 no.6
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• pp.315-323
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• 2009

In order to elucidate ultimate biological function of the genome, the model animal system carrying mutations is indispensable. Recently, large-scale mutagenesis projects have been launched in various species. Especially, the mouse is considered to be an ideal model to human because it is a mammalian species accompanied with well-established genetic as well as embryonic technologies. In 1990', large-scale mouse mutagenesis projects firstly initiated with a potent chemical mutagen, N-ethyl-N-nitrosourea (ENU) by the phenotype-driven approach or forward genetics. The knockout mouse mutagenesis projects with trapping/conditional mutagenesis have then followed as Phase II since 2006 by the gene-driven approach or reverse genetics. Recently, the next-generation gene targeting system has also become available to the research community, which allows us to establish and analyze mutant mice carrying an allelic series of base substitutions in target genes as another reverse genetics. Overall trends in the large-scale mouse mutagenesis will be reviewed in this article particularly focusing on the new advancement of the next-generation gene targeting system. The drastic expansion of the mutant mouse resources altogether will enhance the systematic understanding of the life. The construction of the mutant mouse resources developed by the forward and reverse genetic mutagenesis is just the beginning of the annotation of mammalian genome. They provide basic infrastructure to understand the molecular mechanism of the gene and genome and will contribute to not only basic researches but also applied sciences such as human disease modelling, genomic medicine and personalized medicine.

• BMB Reports
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• v.37 no.1
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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.

• Korean Journal of Cleft Lip And Palate
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• v.11 no.2
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• pp.65-70
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• 2008

Cleft lip and/or palate are common birth defects in humans and the causes including multiple genetic and environmental factors are complex. Combinations of genetic, biochemical, and embryological approaches in the laboratory mice are used to investigate the molecular mechanisms underlying normal craniofacial development and the congenital craniofacial malformations including cleft lip and/or palate. Both forward and reverse genetic approaches are used. The forward genetic approach involves identification of causative genes and molecular pathways disrupted by uncharacterized mutations that cause craniofacial malformations including cleft lip and/or cleft palate. The reverse genetic approach involves generation and analyses of mice carrying null or conditional mutations using the Cre-loxP mediated gene targeting techniques.

• Reproductive and Developmental Biology
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• v.38 no.3
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• pp.99-105
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• 2014

In this study, to further understand the mechanism of animal growth and to develop a miniature transgenic animal model, we constructed and tested tetracycline-inducible RNAi system using shRNA targeting the mRNA of mouse insulin-like growth factor (mIGF-1) or mouse growth hormone receptor (mGHR) gene. Quantitative real-time PCR analysis of mouse liver cell (Hepa1c1c7) cells transfected with these vectors showed 85% or 90% of expression inhibition effect of IGF-1 or GHR, respectively. In ELISA analysis, the protein level of IGF-1 in the cells expressing the shRNA targeting IGF-1 mRNA was reduced to 26% of non-transformed control cells. Unexpectedly, in case of using shRNA targeting GHR, the IGF-1 protein level was decreased to 75% of control cells. Further experiments are needed to explain the lower interference effect of GHR shRNA in IGF-1 protein. Accumulated knowledge of this approach could be applicable to a variety of related biological area including gene function study, gene therapy, development of miniature animals, etc.

• BMB Reports
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• v.52 no.1
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• pp.70-85
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• 2019

Reduction of insulin/insulin-like growth factor 1 (IGF1) signaling (IIS) extends the lifespan of various species. So far, several longevity mouse models have been developed containing mutations related to growth signaling deficiency by targeting growth hormone (GH), IGF1, IGF1 receptor, insulin receptor, and insulin receptor substrate. In addition, p70 ribosomal protein S6 kinase 1 (S6K1) knockout leads to lifespan extension. S6K1 encodes an important kinase in the regulation of cell growth. S6K1 is regulated by mechanistic target of rapamycin (mTOR) complex 1. The v-myc myelocytomatosis viral oncogene homolog (MYC)-deficient mice also exhibits a longevity phenotype. The gene expression profiles of these mice models have been measured to identify their longevity mechanisms. Here, we summarize our knowledge of long-lived mouse models related to growth and discuss phenotypic characteristics, including organ-specific gene expression patterns.

• Journal of Microbiology and Biotechnology
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• v.19 no.9
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• pp.1070-1076
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• 2009

Telomerase reverse transcriptase (TERT), which prolongs the replicative life span of cells, is highly upregulated in 85-90% of human cancers, whereas most normal somatic tissues in humans express limited levels of the telomerase activity. Therefore, TERT has been a potential target for anticancer therapy. Recently, we described a new approach to human cancer gene therapy, which is based on the group I intron of Tetrahymena thermophila. This ribozyme can specifically mediate RNA replacement of human TERT (hTERT) transcript with a new transcript harboring anticancer activity through a trans-splicing reaction, resulting in selective regression of hTERT-positive cancer cells. However, to validate the therapeutic potential of the ribozyme in animal models, ribozymes targeting inherent transcripts of the animal should be developed. In this study, we developed a Tetrahymena-based trans-splicing ribozyme that can specifically target and replace the mouse TERT (mTERT) RNA. This ribozyme can trigger transgene activity not only also in mTERT-expressing cells but hTERT-positive cancer cells. Importantly, the ribozyme could selectively induce activity of the suicide gene, a herpes simplex virus thymidine kinase gene, in cancer cells expressing the TERT RNA and thereby specifically hamper the survival of these cells when treated with ganciclovir. The mTERT-targeting ribozyme will be useful for evaluation of the RNA replacement approach as a cancer gene therapeutic tool in the mouse model with syngeneic tumors.

• Reproductive and Developmental Biology
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• v.29 no.1
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• pp.19-24
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• 2005

The low density lipoprotein receptor-related protein 5 (LRP5) highly expressed in many tissues, including hepatocytes and pancreatic beta cells, can bind to apolipoprotein E. To evaluate in vivo roles of LRP5, we generated LRP5-deficient mice. LRP5 genomic DNA was isolated from TT2 embryonic stem (ES) cells. Targeting vector was constructed to disrupt an exon 18 of the mouse LRP5 gene and transfected into ES cells. Three homologous recombinants at LRP5 locus were identified from 178 G418-resistant clones. Chimeric males generated by morula aggregation technique were mated to C57BL/6 female mice. After achieving germ-line transmission, LRP5+/- females were crossed with LRP5+/- males to obtain LRP5-deficient mice. One line of mice lacking LRP5 gene was confirmed by Southern blotting. Such knock-out mice may serve as an effective animal model to study in vivo function of LRP5 gene.

• Journal of Veterinary Science
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• v.21 no.2
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• pp.26.1-26.14
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• 2020

Pancreatic ductal adenocarcinoma is a lethal cancer type that is associated with multiple gene mutations in somatic cells. Genetically engineered mouse is hardly applicable for developing a pancreatic cancer model, and the xenograft model poses a limitation in the reflection of early stage pancreatic cancer. Thus, in vivo somatic cell gene engineering with clustered regularly interspaced short palindromic repeats is drawing increasing attention for generating an animal model of pancreatic cancer. In this study, we selected Kras, Trp53, Ink4a, Smad4, and Brca2 as target genes, and applied Campylobacter jejuni Cas9 (CjCas9) and Streptococcus pyogens Cas9 (SpCas9) for developing pancreatic cancer using adeno associated virus (AAV) transduction. After confirming multifocal and diffuse transduction of AAV2, we generated SpCas9 overexpression mice, which exhibited high double-strand DNA breakage (DSB) in target genes and pancreatic intraepithelial neoplasia (PanIN) lesions with two AAV transductions; however, wild-type (WT) mice with three AAV transductions did not develop PanIN. Furthermore, small-sized Cjcas9 was applied to WT mice with two AAV system, which, in addition, developed high extensive DSB and PanIN lesions. Histological changes and expression of cancer markers such as Ki67, cytokeratin, Mucin5a, alpha smooth muscle actin in duct and islet cells were observed. In addition, the study revealed several findings such as 1) multiple DSB potential of AAV-CjCas9, 2) peri-ductal lymphocyte infiltration, 3) multi-focal cancer marker expression, and 4) requirement of > 12 months for initiation of PanIN in AAV mediated targeting. In this study, we present a useful tool for in vivo cancer modeling that would be applicable for other disease models as well.

• Journal of Life Science
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• v.22 no.10
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• pp.1428-1433
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• 2012

Longevity is an exciting but difficult subject to study because it is determined by complex processes that require the coordinated action of several genetic factors as well as physiological and environmental influences. Genetic approaches have been applied to animal models to identify the molecular mechanism responsible for longevity. Several experimental model organisms obtained over the last decades suggest that the complete deletion of a single gene by gene targeting has proven to be an invaluable tool for the discovery of the mechanisms underlying longevity. The first discovery of long-lived mutants came from Caenorhabditis elegans research, which identified the insulin/IGF-1 pathway as responsible for longevity in this worm. IGF-1 is a multifunctional polypeptide that has sequence similarity to insulin and is involved in normal growth and development of cells. Several factors in the IGF-1 system have since been studied by gene targeting in the control of longevity in lower species, including nematode and fruit fly. In addition, significant progress has been made using mice models to extend the lifespan by targeted mutations that interfere with growth hormone/IGF-1 and IGF-1 signaling cascades. A recent finding that IGF-1 is involved in aging in mice was achieved by using liver-specific knockout mutant mice, and this clearly demonstrated that the IGF-1 signal pathway can extend the lifespan in both invertebrates and vertebrate models. Although the underlying molecular mechanisms for the control of longevity are not fully understood, it is widely accepted that reduced IGF-1 signaling plays an important role in the control of aging and longevity. Several genes involved in the IGF-1 signaling system are reviewed in relation to longevity in genetically modified mice models.