• Journal of Life Science
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• v.17 no.4 s.84
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• pp.587-592
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• 2007

Manipulation of the mouse genome by activating or inactivating the gene has contributed to the understanding of the function of the gene in the subset of cells during embryonic development or postnatal period of life. Most of all, gene targeting, which largely depends on the availability of mouse embryonic stem (ES) cells, is the milestone of development of animal models for human disease. Recombinase-mediated genome modification (Cre-LoxP and Flp-Frt etc) and the ligand-dependent regulation system, more accurate and elaborate manipulation tools, have been successfully developed and applied to dissect the mechanisms governing complex biological processes and to understand the role of protein in temporal-and spatial aspects of development. As technologies concerning refined manipulation of mouse genome are developed, they are expected to open new opportunities to better understand the diverse in vivo functions of genes.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.5
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• pp.2409-2414
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• 2012

Objective: Traditional Chinese herbal medicines have a very long history. Rosa roxburghii Tratt and Fagopyrum cymosum are two examples of plants which are reputed to have benefits in improving immune responses, enhancing digestive ability and demonstrating anti-aging effects. Some evidence indicates that herbal medicine soups containing extracts from the two in combination have efficacy in treating malignant tumors. However, the underlying mechanisms are far from well understood. The present study was therefore undertaken to evaluate anticancer effects and explore molecular mechanisms in vitro. Methods: Proliferation and apoptosis were assessed with three carcinoma cell lines (human esophageal squamous carcinoma CaEs-17, human gastric carcinoma SGC-7901 and pulmonary carcinoma A549) by MTT assay and flow cytometry, respectively, after exposure to extract from Rosa roxburghii Tratt (CL) and extract from Fagopyrum cymosum (FR). $IC_{30}$ of CL and FR were obtained by MTT assay. Tumor cells were divided into four groups : control with no exposure to CL or FR; CL with $IC_{30}$ CL; FR with $IC_{30}$ FR; CL+FR group with 1/2 ($IC_{30}$ CL + $IC_{30}$ FR). RT-PCR and Western blot analysis were used to detect the expression of Ki-67, Bax and Bcl-2 at mRNA and protein levels. Results: Compared with the CL or FR groups, the combination of CL+FR showed significant inhibition of cell growth and increase in apoptosis; the mRNA and protein expression levels of Ki-67 and Bcl-2 in CL+FR group were all greatly decreased, while the expression of Bax was markedly increased. Conclusions: These results indicate that the synergistic antitumor effects of combination of CL and FR are related to inhibition of proliferation and induction of apoptosis.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.4
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• pp.311-319
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• 2009

Objective: Human embryonic stem cells (hESCs) have the capacity to differentiate into all of the cell types and therefore hold promise for cell therapeutic applications. In order to utilize this important potential of hESCs, enhancement of currently used technologies for handling and manipulating the cells is required. The cryopreservation of hESC colonies was successfully performed using the vitrification and slow freezing-rapid thawing method. However, most of the hESC colonies were showed extremely spontaneous differentiation after freezing and thawing. In this study, we were performed to rapidly collect of early passage hESCs, which was thawed and had high rate of spontaneously differentiation of SNUhES11 cell line. Methods: Four days after plating, partially spontaneously differentiated parts of hESC colony were cut off using finely drawn-out dissecting pipette, which is mechanical separation method. Results: After separating of spontaneously differentiated cells, we observed that removed parts were recovered by undifferentiated cells. Furthermore, mechanical separation method was more efficient for hESCs expansion after thawing when we repeated this method. The recovery rate after removing differentiated parts of hESC colonies were 55.0%, 74.5%, and 71.1% when we have applied this method to three passages. Conclusion: Mechanical separation method is highly effective for rapidly collecting and large volumes of undifferentiated cells after thawing of cryopreserved early passage hESCs.

• Laboraroty Animal Research
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• v.34 no.4
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• pp.166-175
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• 2018

Recombination activating gene-2 (RAG-2) plays a crucial role in the development of lymphocytes by mediating recombination of T cell receptors and immunoglobulins, and loss of RAG-2 causes severe combined immunodeficiency (SCID) in humans. Rag-2 knockout mice created using homologous recombination in ES cells have served as a valuable immunodeficient platform, but concerns have persisted on the specificity of Rag-2-related phenotypes in these animals due to the limitations associated with the genome engineering method used. To precisely investigate the function of Rag-2, we recently established a new Rag-2 knockout FVB mouse line ($Rag-2^{-/-}$) manifesting lymphopenia by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease. In this study, we further characterized their phenotypes focusing on histopathological analysis of lymphoid organs. $Rag-2^{-/-}$ mice showed no abnormality in development compared to their WT littermates for 26 weeks. At necropsy, gross examination revealed significantly smaller spleens and thymuses in $Rag-2^{-/-}$ mice, while histopathological investigation revealed hypoplastic white pulps with intact red pulps in the spleen, severe atrophy of the thymic cortex and disappearance of follicles in lymph nodes. However, no perceivable change was observed in the bone marrow. Moreover, our analyses showed a specific reduction of lymphocytes with a complete loss of mature T cells and B cells in the lymphoid organs, while natural killer cells and splenic megakaryocytes were increased in $Rag-2^{-/-}$ mice. These findings indicate that our $Rag-2^{-/-}$ mice show systemic lymphopenia with the relevant histopathological changes in the lymphoid organs, suggesting them as an improved Rag-2-related immunodeficient model.

• Laboraroty Animal Research
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• v.34 no.4
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• pp.257-263
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• 2018

Trefoil factor 1 (TFF1, also known as pS2) is strongly expressed in the gastrointestinal mucosa and plays a critical role in the differentiation of gastric glands. Since approximately 50% of all human gastric cancers are associated with decreased TFF1 expression, it is considered a tumor suppressor gene. Tff1 deficiency in mice results in histological changes in the antral and pyloric gastric mucosa, with severe hyperplasia and dysplasia of epithelial cells, resulting in the development of antropyloric adenoma. Here, we generated Tff1-knockout (KO) mice, without a neomycin resistant ($Neo^R$) cassette, using the clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRSIPR/Cas9) system. Though our Tff1-KO mice showed phenotypes very similar to the previous embryonic stem (ES)-cell-based KO mice, they differed from the previous reports in that a reduction in body weight was observed in males. These results demonstrate that these newly established Tff1-KO mice are useful tools for investigating genetic and environmental factors influencing gastric cancer, without the effects of artificial gene insertion. Furthermore, these findings suggest a novel hypothesis that Tff1 expression influences gender differences.