• Toxicological Research
• /
• v.24 no.1
• /
• pp.69-78
• /
• 2008

Mutant mouse which show dwarfism has been developed by N-ethyl-N-nitrosourea (ENU) mutagenesis using BALB/c mice. The mutant mouse was inherited as autosomal recessive trait and named Small Body Size (SBS) mouse. The phenotype of SBS mouse was not apparent at birth, but it was possible to distinguish mutant phenotype from normal mice 1 week after birth. In this study, we examined body weight changes and bone mineral density (BMD), and we also carried out genetic linkage analysis to map the causative gene(s) of SBS mouse. Body weight changes were observed from birth to 14 weeks of age in both affected (n = 30) and normal mice (n = 24). BMD was examined in each five SBS and normal mice between 3 and 6 weeks of age, respectively. For the linkage analysis, we produced backcross progeny [(SBS${\times}$C57BL/6J) $F_1{\times}$ SBS] $N_2$ mice (n = 142), and seventy-four microsatellite markers were used for primary linkage analysis. Body weight of affected mice was consistently lower than that of the normal mice, and was 43.7% less than that of normal mice at 3 weeks of age (P < 0.001). As compared with normal mice at 3 and 6 weeks of age, BMD of the SBS mice was significantly low. The results showed 15.5% and 14.1 % lower in total body BMD, 15.3% and 8.7% lower in forearm BMD, and 29.7% and 20.1% lower in femur BMD, respectively. The causative gene was mapped on chromosome 10. The map order and the distance between markers were D10Mit248 - 2.1 cM - D10Mit51 - 4.2 cM - sbs - 0.7 cM - D10Mit283 - 1.4cM - D10Mit106 - 11.2cM - D10Mit170.

• Proceedings of the KSAR Conference
• /
• 2003.06a
• /
• pp.80-80
• /
• 2003

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor, cognitive, and psychiatric symptoms, accompanied by marked cell death in the striatum and cortex. Stereotaxic injection of quinolinic acid (QA) into striatum results in a degeneration of GABAergic neurons and exhibits abnormal motor behaviors typical of the illness. The objective of this study was carried out to obtain basic information about whether parthenogenetic mouse embryonic stem (PmES) cells are suitable for cell replacement therapy of HD. To establish PmES cell lines, hybrid F1 (C57BL/6xCBA/N) mouse oocytes were treated with 7% ethanol for 5 min and cytochalasin-B for 4 hr to initiate spontaneous cleavage. Thus established PmES cells were induced to differentiate using bFGF (20ng/ml) followed by selection of neuronal precursor cells for 8 days in N2 medium. After selection, cells were expanded at the presence of bFGF (20 ng/ml) for another 6 days, then a final differentiation step in N2 medium for 7 days. To establish recipient animal models of HD, young adult mice (7 weeks age ICR mice) were lesioned unilaterally with a stereotaxic injection of QA (60 nM) into the striatum and the rotational behavior of the animals was tested using apomorphine (0.1mg/kg, IP) 7 days after the induction of lesion. Animals rotating more than 120 turns per hour were selected and the differentiated PmES cells (1$\times$10$^4$cells/ul) were implanted into striatum. Four weeks after the graft, immunohistochemical studies revealed the presence of cells reactive to anti-NeuN antibody. However, only a slight improvement of motor behavior was observed. By Nissl staining, cell mass resembling tumor was found at the graft site and near cortex which may explain the slight behavioral improvement. Detailed experiment on cell viability, differentiation and migration explanted in vivo is currently being studied.

• Molecules and Cells
• /
• v.25 no.3
• /
• pp.358-367
• /
• 2008

The embryonic germ cell (EGCs) of mice is a kind of pluripotent stem cell that can be generated from pre- and post-migratory primordial germ cells (PGCs). Most previous studies on DNA methylation of EGCs were restricted to 12.5 days post coitum (dpc). This study was designed to establish and characterize murine EGC lines from migrated PGCs as late as 13.5 dpc and to estimate the degrees of methylation of their imprinted genes as well as of the non-imprinted locus, Oct4, using an accurate and quantitative method of measurement. We established five independent EGC lines from post migratory PGCs of 11.5-13.5 dpc from C57BL/6 ${\times}$ DBA/2 F1 hybrid mouse fetuses. All the EGCs exhibited the typical features of pluripotent cells including hypomethylation of the Oct4 regulatory region. We examined the methylation status of three imprinted genes; Igf2, Igf2r and H19 in the five EGC lines using bisulfite genomic sequencing analysis. Igf2r was almost unmethylated in all the EGC lines irrespective of the their sex and stage of isolation; Igf2 and H19 were more methylated than Igf2r, especially in male EGCs. Moreover, EGCs derived at 13.5 dpc exhibited higher levels of DNA methylation than those from earlier stages. These results suggest that in vitro derived EGCs acquire different epigenotypes from their parental in vivo migratory PGCs, and that sex-specific de novo methylation occurs in the Igf2 and H19 genes of EGCs.

• The Korea Journal of Herbology
• /
• v.24 no.3
• /
• pp.13-19
• /
• 2009

Objectives : This study was aimed to define the protective effect of Tissue Cultured Root of Wild Ginseng (CWG) against doxorubicin (Doxo) toxicity, and investigate the anti-tumor synergic effect of CWG in combination with Doxo in tumor-bearing C57BL/6 mice. Methods : Tumor-bearing mice were established by single inoculation with B16/F10 melanoma cells (2$\times$10$^6$/ml) subcutaneously. Tumor-bearing mice (tumor volume between 50-100 mm$^3$) were selected and divided them into control, Doxo, and Doxo+CWG group. Mice of Doxo group were received with Doxo (4 mg/kg of B.W.) intraperitoneally at 0, 4, 8 days after starting the experiment. Mice of Doxo+CWG group were received CWG water extract during 12 days in combination with Doxo treatment. The body weight, tumor volume, tumor weight, and organ weight (heart, liver, kidney, and testis) were measured. And serum SPK, GOT and creatinine values were analysed. Results : The volume and weights of tumor masses in Doxo group were decreased significantly compared with the those of control group. And the those of Doxo+CWG group were not significantly different from the those of Doxo group. Whereas the weight of body, liver, kidney and testis in Doxo+CWG group were increased significantly compared with the those of Doxo group. The level of serum CPK and GOT in Doxo group were increased compared with the those of control group. But the value of Doxo+CWG group were decreased significantly compared with the values of Doxo group. Conclusions : These results suggest that CWG has protective effect against doxorubicin toxicity. And these effect is guessed that is caused in augmentation of vital energy.

• The Korean Journal of Physiology and Pharmacology
• /
• v.21 no.3
• /
• pp.317-325
• /
• 2017

Non-alcoholic fatty liver disease (NAFLD) has become the most prevalent liver disease in parallel with worldwide epidemic of obesity. Reactive oxygen species (ROS) contributes to the development and progression of NAFLD. Peroxisomes play an important role in fatty acid oxidation and ROS homeostasis, and catalase is an antioxidant exclusively expressed in peroxisome. The present study examined the role of endogenous catalase in early stage of NAFLD. 8-week-old male catalase knock-out (CKO) and age-matched C57BL/6J wild type (WT) mice were fed either a normal diet (ND: 18% of total calories from fat) or a high fat diet (HFD: 60% of total calories from fat) for 2 weeks. CKO mice gained body weight faster than WT mice at early period of HFD feeding. Plasma triglyceride and ALT, fasting plasma insulin, as well as liver lipid accumulation, inflammation (F4/80 staining), and oxidative stress (8-oxo-dG staining and nitrotyrosine level) were significantly increased in CKO but not in WT mice at 2 weeks of HFD feeding. While phosphorylation of Akt (Ser473) and $PGC1{\alpha}$ mRNA expression were decreased in both CKO and WT mice at HFD feeding, $GSK3{\beta}$ phosphorylation and Cox4-il mRNA expression in the liver were decreased only in CKO-HF mice. Taken together, the present data demonstrated that endogenous catalase exerted beneficial effects in protecting liver injury including lipid accumulation and inflammation through maintaining liver redox balance from the early stage of HFD-induced metabolic stress.

• The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
• /
• v.15 no.2
• /
• pp.80-103
• /
• 2002

To screen the effective materials for hair loss treatment, several natural extracts were tested using in vivo and in vitro test models. Firstly, all test materials were applicated onto the back skin of C57BL／6 mouse and then hair growth pormoting effect were measured using hair growth index As a result, Polygonum muitifiorum Thunb and Terrninalia chebula Retz. showed potent hair growth promoting effect, ranking as 1.5-2.0 of hair growth index. However, there were no plant extracts, which have remarkable potential of growth promotion of human hair dermal papilla cells cultured in vitro. In the experiments of 5${\alpha}$-reductase type Ⅱ inhibition assay, Morus alba L., Chaenomelis Fructus, Saussureae Radix, Angelicae Gigantis Radix, Polygonum multifiorum Thunb, and Angelica dahurica (Fischer) Bentham et Hooker f. showed effective potential to inhibit the activity of 5${\alpha}$-reductase type Ⅱ. To investigate the possible involvement of effects of several plant extracts on the gene expression of growth factors in human hair dermal papilla cells, RT-PCR analyses were performed. As a consequences, Mentha haplocalyx Briq., Cimicifuga foetida L., Eclipta prostrata (L.) L., Pinus densiflora S. et. Z, and Polygonum muitifiorum Thunb revealed the regulatory roles on the expression of growth factors such as IGF-I, KGF, HGF and VEGF in the dermal papilla cells. Another test for inhibition of microbial such as P. acne and P. ovale were also carried out to find whether these plant extracts have anti-microbial activities. Morus alba L. and Chaenomelis Fructus showed anti-microbial effects on Propionibacterium acnes, which is believed as a pathogen of acne. Together, these results showed several plant extracts can be used for hair growth promotion.

• Clinical and Experimental Reproductive Medicine
• /
• v.29 no.2
• /
• pp.129-138
• /
• 2002

Objective: This study was to compare the characteristics between parthenogenetic mES (P-mES) cells and in vitro fertilization mES cells. Materials and Methods: Mouse oocytes were recovered from superovulated 4 wks hybrid F1 (C57BL/6xCBA/N) female mice. For parthenogenetic activation, oocytes were treated with 7% ethanol for 5 min and $5{\mu}g$/ml cytochalasin-B for 4 h. For IVF, oocytes were inseminated with epididymal sperm of hybrid F1 male mice ($1{times}10^6/ml$). IVF and parthenogenetic embryos were cultured in M16 medium for 4 days. Cell number count of blastocysts in those two groups was taken by differential labelling using propidium iodide (red) and bisbenzimide (blue). To establish ES cells, b1astocysts in IVF and parthenogenetic groups were treated by immunosurgery and recovered inner cell mass (ICM) cells were cultured in LIF added ES culture medium. To identify ES cells, the surface markers alkaline phosphatase, SSEA-1, 3,4 and Oct4 staining were examined in rep1ated ICM colonies. Chromosome numbers in P-mES and mES were checked. Also, in vitro differentiation potential of P-mES and mES was examined. Results: Although the cleavage rate (${\geq}$2-cell) was not different between IVF (76.3%) and parthenogenetic group (67.0%), in vitro development rate was significantly low in parthenogenetic group (24.0%) than IVF group (68.4%) (p<0.05). Cell number count of ICM and total cell in parthenogenetic b1astocysts ($9.6{\pm}3.1,\;35.1{\pm}5.2$) were signficantly lower than those of IVF blastocysts ($19.5{\pm}4.7,\;63.2{\pm}13.0$) (p<0.05). Through the serial treatment procedure such as immunosurgery, plating of ICM and colony formation, two ICM colonies in IVF group (mES, 10.0%) and three ICM colonies (P-mES, 42.9%) in parthenogenetic group were able to culture for extended duration (25 and 20 passages, respectively). Using surface markers, alkaline phosphatase, SSEA-l and Oct4 in P-mES and mES colony were positively stained. The number of chromosome was normal in ES colony from two groups. Also, in vitro neural and cardiac cell differentiation derived from mES or P-mES cells was confirmed. Conclusion: This study suggested that P-mES cells can be successfully established and that those cell lines have similar characteristics to mES cells.

• Clinical and Experimental Reproductive Medicine
• /
• v.16 no.2
• /
• pp.139-146
• /
• 1989

The purpose of this study was to examine the qualitative variation of human fetal-cord sera (HCS) and to accept the sera in human lVF-ET program. One hundred and sixteenth RCS were tested with 1772 2-cell embryos of F1 (C57BL x CBA) virgin mice, Ten to sixteenth embryos were cultured in m-KRB medium with a aliquot of each serum (10%, v/v) or with bovine serum albumin(O.4%, w/v) as a control medium. Embryonic development were recorded at every 24hr for 4 days by such events as cellular compaction, cavitation, and hatching. In the control groups of eight assays, 98.1%(106/ 108) of 2-ce1l embryos developed above expanded blastocyst and the embryonic development was unified through the tests. But the developmental pattern in medium with each serum was various. Namely, the sera that supported development of 100% 2-cell embryos to above morula, early blastocyst, expanded blastocyst and hatching blastocyst was 45,7%(53/116) , 35.3%(41/116), 15.5%08/116.) and 6.9-%(8/116), respectively. And the sera that supported development of above 80% 2-cell embryos to the each embryonic stage was 92.2% (107/116), 83.6%(97/116), 63.8%(74/116) and 36.2%(42/116), respectively. Meanwhile two kinds of toxic pattern to the embryonic development were observed in some sera. The first pattern is that some sera arrested development of most embryos in pre- or post-stage of morula or blastocyst. The second pattern is that some sera promoted or arrested a part of embryos in the same dish. The ability of serum was depended on the batch of serum. Finally we could accept 69%(80/116) of the tested sera for human IVF-ET program. The base line for acceptance was the ability that supported above 80% 2-ce1l embryos to blastocyst. But some deterious sera were contained in this range. We cut off about 10% of the sera (83.6% , 97/116) that passed the baseline. This final percent of sera was similar to that of grade N of this study.

• Journal of Pharmacopuncture
• /
• v.6 no.1
• /
• pp.76-94
• /
• 2003

Objectives : The purpose of this study was to investigate Acute$\cdot$Subacute Toxicity and Anti-cancer Effect of K-Herbal-acupuncture in mice and rats. Methods : Balb/c mice were injected intraperitoneally with K-herbal-acupuncture for $LD_{50}$ and acute toxicity test. Sprague-Dawley rats were injected intraperitoneally with K-herbal-acupuncture for subacute toxicity test. K-Herbal-acupuncture was injected on abdomen of mice with S-180 cancer cell line. Result : 1. $LD_{50}$ of K-Herbal-acupuncture was limited $4{\times}10^{-3}$ml/kg~$2{\times}10^{-3}$ml/kg by the test. 2. In acute toxicity test, all of mice were down to the moving reflex, but the weight of mice was increased in treatment group, compared with the normal group. (P<0.05) 3. In acute toxicity test of serum biochemical values of mice, glucose was increased in treatment II group, total cholesterol was increased both treatments.(P<0.05) 4. In subacute toxicity test, the clinical signs of toxication was down to the moving reflex, but it is not severe like acute toxicity test, and observed weight loss at the treatments. 5. In subacute toxicity test, liver weight was decreased compared with the normal group. (P<0.05) 6. In subacute toxicity test of complete blood count test (CBC) of rat, HCT was decreased in treatments, compared with the normal group.(P<0.05) 7. In subacute toxicity test of serum biochemical values of rat, uric acid and triglyceride were decreased, and glucose was increased in treatment groups compared with the control group. (P<0.05) 8. Median survival time was increased about $45\%$ in treatment groups compared with the control group.(P<0.05) 9. Natural killer cell activity was increased in B16F10 lung cancer model, but it was not in sarcoma-180 abdomen cancer. 10. In interleukin-2 productivity test, treatment groups didn't show significant change in lung cancer and abdomen cancer, compared with the normal group.(P<0.005) 11. In making an examination of metastatic cancer with the naked eye, melanoma metastasized in the Lung of C57BL/6 mice. The treated group showed more Melanoma than the control in the numbers and volume. Conclusion : According to the result, K-herbal-acupuncture need further study to know the function and effect in cancer.

• Korean Journal of Animal Reproduction
• /
• v.25 no.2
• /
• pp.163-169
• /
• 2001

OSBA(oocytes-sperm binding assay) is a tool developed for rapid test of optimal condition of IVF medium and protein source by binding ability of mouse sperm and egg. Mouse oocyte-cumulus complexes were prepared by removing of the cumulus cells with 0.1% hyaluronidase. 10$\pm$2 oocytes per 30 ${mu}ell$ medium drop were inseminated with 3 ${mu}ell$ sperm suspension and were cultured f3r 3 hours and 24 hours, respectively. And the oocytes were recovered gently and the No. of sperm bound on oocytes were counted. In the Exp. 1, the ratio of oocytes bound with one sperm at least were 60.2%(50/83), 2%(2/77) and 100%(79/79) in the medium with no protein, FBS(15%, v/v) and BSA(0.4%. w/v), respectively, Fetal bovine serum(FBS) seriously inhibited sperm binding on oocyte, although bovine serum albumin(BSA) promoted the binding ability. The inhibiting effect of FBS was dependent on the concentration of FBS. The sperm binding ability according to oocyte maturity was tested in the Exp. 2. There was no significant difference between Met. II (mature) and Met. I (intermediate mature) oocytes in the number of oocytes bound with sperm and the number of sperm bound on oocytes. Finally, in Exp. 3, two batches of Ham's F10 medium with good and poor quality by OSBA were tested (The ratios of embryos developed from PN 1-cell stage to hatched blastocyst; 25% vs. 70%). In the medium with good quality, sperm binding ability was significantly increased (P ＜ 0.05). The ratio of oocytes bound with one sperm at least was 66% and 90% in the medium with poor and good quality, respectively. Conclusively, It was possible to test IVF medium condition rapidly and easily by OSBA.