Use of laboratory animals and issues regarding the procurement of animals for research in Korea / 한국실험동물학회지

Background@#Laboratory animals remain critical to biomedical research, despite the increasing availability of alternative approaches. Indeed, scientists strive to reduce and refine and replace the use of laboratory animals, even in the face of public calls for ever-more stringent regulation for the protection and care of animals in research. This report outlines the current status and legal regulatory issues with regard to the procurement and use of animals for research in Korea. @*Results@#The number of animals used for education and research purposes was increased nationwide, from 2.5 to 4.9 million in 2015 and 2021, respectively. When compared with figures from the UK, institutions in Korea were found to use more mammals such as mice and dogs. In our research, we identified three major issues concerning recent animal supply in Korea, particularly: (1) Purchase of dogs from unregistered animal supplier for a dog cloning project; (2) Purchase of dogs from an unclear source for veterinary education and training; (3) Illegal cat experiments using cats obtained from unauthorized routes. @*Conclusions@#Our findings support the notion that alternatives to laboratory animal research should be implemented. We conclude that improvements in the regulations and guidelines for animal suppliers, together with the recent introduction of legislation will improve animal safety and wellbeing of animals in laboratory research in Korea.

Operational issues of the Institutional Animal Care and Use Committee in Korea

Korean Institutional Animal Care and Use Committee (IACUC) is currently facing some operational pressing issues. 1) Review of the animal protocol containing controversial technology. 2) Review of the multi-institution animal protocol. 3) Review of veterinary clinical trials for client-owned animals. 4) Delay in the review process in large institutions with a single IACUC. Here, the following three solutions are proposed to address the above issues. 1) Establishment of public IACUC. 2) Establishment of the Veterinary Clinical Study Committee as an advisory body to the IACUC. 3) Operating multiple committees rather than increasing the number of committee members on a single committee.

Expression patterns of Rho-associated protein kinase signaling pathway-related genes in mouse submandibular glands

Salivary glands are exocrine glands that secrete saliva into the oral cavity, and secreted saliva plays essential roles in oral health. Therefore, maintaining the salivary glands in an intact state is required for proper production and secretion of saliva. To investigate a specific signaling pathway that might affect the maintenance of mouse submandibular gland (SMGs), RNA sequencing was performed. In SMGs, downregulated expression patterns of Rho-associated protein kinase (ROCK) signaling pathway-related genes, including Rhoa, Rhob, Rhoc, Rock1, and Rock2, were observed. Gene expression profiling analyses of these genes indicate that the ROCK signaling pathway is a potential signal for SMG maintenance.

Expression patterns of Rho-associated protein kinase signaling pathway-related genes in mouse submandibular glands

Salivary glands are exocrine glands that secrete saliva into the oral cavity, and secreted saliva plays essential roles in oral health. Therefore, maintaining the salivary glands in an intact state is required for proper production and secretion of saliva. To investigate a specific signaling pathway that might affect the maintenance of mouse submandibular gland (SMGs), RNA sequencing was performed. In SMGs, downregulated expression patterns of Rho-associated protein kinase (ROCK) signaling pathway-related genes, including Rhoa, Rhob, Rhoc, Rock1, and Rock2, were observed. Gene expression profiling analyses of these genes indicate that the ROCK signaling pathway is a potential signal for SMG maintenance.

GRIM-19 Ameliorates Multiple Sclerosis in a Mouse Model of Experimental Autoimmune Encephalomyelitis with Reciprocal Regulation of IFNγ/Th1 and IL-17A/Th17 Cells

The protein encoded by the Gene Associated with Retinoid-Interferon-Induced Mortality-19 (GRIM-19) is located in the mitochondrial inner membrane and is homologous to the NADH dehydrogenase 1-alpha subcomplex subunit 13 of the electron transport chain.Multiple sclerosis (MS) is a demyelinating disease that damages the brain and spinal cord.Although both the cause and mechanism of MS progression remain unclear, it is accepted that an immune disorder is involved. We explored whether GRIM-19 ameliorated MS by increasing the levels of inflammatory cytokines and immune cells; we used a mouse model of experimental autoimmune encephalomyelitis (EAE) to this end. Six-to-eight-week-old male C57BL/6, IFNγ-knockout (KO), and GRIM-19 transgenic mice were used; EAE was induced in all strains. A GRIM-19 overexpression vector (GRIM19 OVN) was electrophoretically injected intravenously. The levels of Th1 and Th17 cells were measured via flow cytometry, immunofluorescence, and immunohistochemical analysis. IL-17A and IFNγ expression levels were assessed via ELISA and quantitative PCR. IL-17A expression decreased and IFNγ expression increased in EAE mice that received injections of the GRIM19 OVN. GRIM-19 transgenic mice expressed more IFNγ than did wild-type mice; this inhibited EAE development. However, the effect of GRIM-19 overexpression on the EAE of IFNγ-KO mice did not differ from that of the empty vector. GRIM-19 expression was therapeutic for EAE mice, elevating the IFNγ level. GRIM-19 regulated the Th17/Treg cell balance.

GRIM-19 Ameliorates Multiple Sclerosis in a Mouse Model of Experimental Autoimmune Encephalomyelitis with Reciprocal Regulation of IFNγ/Th1 and IL-17A/Th17 Cells

The protein encoded by the Gene Associated with Retinoid-Interferon-Induced Mortality-19 (GRIM-19) is located in the mitochondrial inner membrane and is homologous to the NADH dehydrogenase 1-alpha subcomplex subunit 13 of the electron transport chain.Multiple sclerosis (MS) is a demyelinating disease that damages the brain and spinal cord.Although both the cause and mechanism of MS progression remain unclear, it is accepted that an immune disorder is involved. We explored whether GRIM-19 ameliorated MS by increasing the levels of inflammatory cytokines and immune cells; we used a mouse model of experimental autoimmune encephalomyelitis (EAE) to this end. Six-to-eight-week-old male C57BL/6, IFNγ-knockout (KO), and GRIM-19 transgenic mice were used; EAE was induced in all strains. A GRIM-19 overexpression vector (GRIM19 OVN) was electrophoretically injected intravenously. The levels of Th1 and Th17 cells were measured via flow cytometry, immunofluorescence, and immunohistochemical analysis. IL-17A and IFNγ expression levels were assessed via ELISA and quantitative PCR. IL-17A expression decreased and IFNγ expression increased in EAE mice that received injections of the GRIM19 OVN. GRIM-19 transgenic mice expressed more IFNγ than did wild-type mice; this inhibited EAE development. However, the effect of GRIM-19 overexpression on the EAE of IFNγ-KO mice did not differ from that of the empty vector. GRIM-19 expression was therapeutic for EAE mice, elevating the IFNγ level. GRIM-19 regulated the Th17/Treg cell balance.

Expression of polo-like kinase 1 in pre-implantation stage murine somatic cell nuclear transfer embryos

Somatic cell nuclear transfer (SCNT) has various applications in research, as well as in the medical field and animal husbandry. However, the efficiency of SCNT is low and the accurate mechanism of SCNT in murine embryo development is unreported. In general, the developmental rate of SCNT murine embryos is lower than in vivo counterparts. In previous studies, polo-like kinase 1 (Plk1) was reported to be a crucial element in cell division including centrosome maturation, cytokinesis, and spindle formation. In an initial series of experiments in this study, BI2536, a Plk1 inhibitor, was treated to in vivo-fertilized embryos and the embryos failed to develop beyond the 2-cell stage. This confirmed previous findings that Plk1 is crucial for the first mitotic division of murine embryos. Next, we investigated Plk1's localization and intensity by immunofluorescence analysis. In contrast to normally developed embryos, SCNT murine embryos that failed to develop exhibited two types of Plk1 expressions; a low Plk1 expression pattern and ectopic expression of Plk1. The results show that Plk1 has a critical role in SCNT murine embryos. In conclusion, this study demonstrated that the SCNT murine embryos fail to develop beyond the 2-cell stage, and the embryos show abnormal Plk1 expression patterns, which may one of the main causes of developmental failure of early SCNT murine embryos.

Effect of the hedgehog signaling pathway on hair formation-related cells

Alopecia has emerged as one of the biggest interests in modern society. Many studies have focused on the treatment of alopecia, such as transplantation of hair follicles or inhibition of the androgen pathway. Hair growth is achieved through proper proliferation of the components such as keratinocytes and dermal papilla cells (DPCs), movement, and interaction between the two cells. The present study examined the effect of the hedgehog (Hh) signaling pathway, which is an important and fundamental signal in the cell, on the morphology and the viability of human keratinocytes and DPCs. Upregulation of Hh signaling caused a morphological change and an increase in epithelium-mesenchymal transition-related gene expression but reduced the viability of keratinocytes, while the alteration of Hh signaling did not cause any change in DPCs. The results show the possibility that the regulation of Hh signaling can be applied for the treatment of alopecia.

The relationship between non-cognitive student attributes and academic achievements in a flipped learning classroom of a pre-dental science course / 한국의학교육

PURPOSE: The purpose of this study was to examine whether non-cognitive student attributes such as learning style and personality type affected academic performance in a flipped learning classroom of a pre-dental undergraduate science course. METHODS: ‘Biodiversity and Global Environment,’ a 15-week, 3-credit course, was designed as a flipped class in Seoul National University School of Dentistry in 2017. Second-year pre-dental students were required to enroll in the course and to engage in online learning and in-class discussion. The Kolb's Learning Style Inventory and the Myers-Briggs Type Indicator were conducted to measure non-cognitive student factors. Independent samples t-test and multivariate regression analyses were used to examine the relationships between self-rated measurements and academic achievement. RESULTS: More than half of the students enrolled in the flipped science course had an assimilator learning style (50%), followed by convergers (24%), accommodators (16%), and divergers (10%), and their personality types were dominated by the introverted, sensing, thinking, and judging types, respectively. Examining group differences using the t-test demonstrated a significant relationship between the diverger group and higher academic success. In particular, the multivariate regression analysis indicated that both thinking types and female students performed better in discussion than feeling types and male students. CONCLUSION: To operate the flipped learning classroom more effectively in medical and dental education, the instructor should carefully develop and apply a more tailored facilitation and relevant assessment by considering student learning styles and personality types.

Flipped-learning course design and evaluation through student self-assessment in a predental science class / 한국의학교육

PURPOSE: This study explores how to design a flipped classroom for a predental science course and evaluate its course through student self-assessment in order to provide practical implications for flipped learning in an undergraduate level. METHODS: Second- and third-year predental students in the Seoul National University School of Dentistry enrolled in Biodiversity and Global Environment, a 15-week, three-credit course based on a flipped learning model. At the end of the course, the students were asked to rate their self-directed learning, attitude toward social media, discussion skills, learning readiness, and class satisfaction. Out of the 82 predental students, 61 (74.3%) answered the survey. Pearson correlation and multivariate regression analyses were employed to examine the relationship between the self-rated measurements and the performance scores. RESULTS: The majority of the students felt somewhat more prepared than the medium level before the class (mean score of 3.17 out of 5.00), whereas they expressed relatively low preference concerning social media use and attitude (mean score of 2.49). Thus, it was found that learning readiness was significantly associated with both discussion skills and class satisfaction. In particular, multivariate regression analysis confirmed that learning readiness had a significant influence on learning outcomes. CONCLUSION: This study offered insights into how to design a flipped learning course in terms of predental students' preference and their learning readiness. Although learning success in a flipped classroom depends on the students' self-perceived level of preparedness, much still remains to be achieved in order to apply social media benefits in a flipped learning context.

Anti-cancer and -Metastatic Effects of Lactobacillus Rhamnosus GG Extract on Human Malignant Melanoma Cells, A375P and A375SM

Human malignant melanoma is an aggressive skin cancer which has been rising at a greater rate than any other cancers. Although various new therapeutic methods have been developed in previous studies, this disease has properties of high proliferation and metastasis rate which remain obstacles that have lead to a poor prognosis in patients. It has been reported that a specific Lactobacillus extract has anti-cancer and –metastasis effect in vitro and in vivo. However, previous research has not specified precisely what effect the Lactobacillus rhamnosus GG (LGG) extract has had on human malignant melanomas. In this study, we showed that the LGG extract has anti-cancer and –metastasis effects on the human malignant melanoma cell lines, A375P and A375SM. At first, it was found that, while the LGG extract affects human neonatal dermal fibroblasts slightly, it induced the dose-dependent anti-cancer effect on A375P and A375SM by a WST-1 proliferation assay. As a result of a real-time PCR analysis, the expression patterns of several genes related to cell cycle, proliferation, and apoptosis were modulating in a manner that inhibited the growth of both malignant melanoma cell lines after the treatment of the LGG extract. Furthermore, genes related to the epithelialmesenchymal transition were down-regulated, and migration rates were also decreased significantly by the LGG extract. Our study showed that the LGG extract could be used as a potential therapeutic source.

Stemness and Proliferation of Murine Skin-Derived Precursor Cells under Hypoxic Environment

Skin-derived precursors (SKPs) have potential to differentiate to various cell types including osteoblasts, adipocytes and neurons. SKPs are a candidate for cell-based therapy since they are easily accessible and have multipotency. Most mammalian cells are exposed to a low oxygen environment with 1 to 5% O2 concentration in vivo, while 21% O2 concentration is common in in vitro culture. The difference between in vitro and in vivo O2 concentration may affect to the behavior of cultured cells. In this report, we investigated the effect of hypoxic condition on stemness and proliferation of SKPs. The results indicated that SKPs exposed to hypoxic condition for 5 days showed no change in proliferation. In terms of mRNA expression, hypoxia maintained expression of stemness markers; whereas, oncogenes, such as Klf4 and c-Myc, were downregulated, and the expression of Nestin, related to cancer migration, was also downregulated. Thus, SKPs cultured in hypoxia may reduce the risk of cancer in SKP cell-based therapy.

Improvement of pregnancy rate by intrauterine administration of dexamethasone and recombinant human leukemia inhibitory factor at the time of embryo transfer in cattle

Bovine embryos (day 5) were cultured to day 10 with or without 100 ng/mL PGF2α in medium supplemented with control; 100 nM Dex; 1,000 U/mL recombinant human leukemia inhibitory factor (rhLIF); or Dex+rhLIF. Although the rates to development to the blastocyst were not significantly different among groups, the hatching rate after additional culture with Dex +/or rhLIF was significantly higher in all supplemented groups than the control (p < 0.05). In the presence of PGF2α, the hatching rate was significantly restored in all supplemented groups relative to the group treated with only PGF2α and the control (p < 0.05). Embryo transfer (ET) was performed with blastocysts (day 7). PGF2α levels of control recipient cows were significantly higher in the circulatory blood samples collected 60 min after ET than in samples collected 60 min before ET (p < 0.005), and were decreased in cows injected with loading medium supplemented with Dex+rhLIF (p < 0.005). Pregnancy rate was significantly higher in the ET group that received supplemented embryo-loading medium than in the non-supplemented control (p < 0.05). The intrauterine administration of Dex and rhLIF at ET prevented increased PGF2α in circulatory blood and resulted in enhanced pregnancy rate.

Comparison of Various Transfection Methods in Human and Bovine Cultured Cells

Transfection is a gene delivery tool that is a popular means of manipulating cellular properties, such as induced pluripotent stem cell (iPSC) generation by reprogramming factors (Yamanaka factors). However, the efficiency of transfection needs to be improved. In the present study, three transfection protocols - non-liposomal transfection (NLT), magnetofection and electroporation - were compared by analysis of their transfection efficiencies and cell viabilities using human dental pulp cells (hDPC) and bovine fetal fibroblasts (bFF) as cell sources. Enhanced green fluorescent protein gene was used as the delivery indicator. For magnetofection, Polymag reagent was administrated. NLT, FuGENE-HD and X-treme GENE 9 DNA transfection reagents were used for NLT. For electroporation, the Neon(TM) and NEPA21(TM) electroporators were tested. Neon(TM) electroporation showed highest transfection efficiency when compared with NLT, magnetofection, and NEPA21(TM) electroporation, with transfection efficiency of about 33% in hDPC and 50% in bFF, based on viable cell population in each cell type. These results suggest that transfection by Neon(TM) electroporation can be used to deliver foreign genes efficiently in human and bovine somatic cells.

A simplified one-step nuclear transfer procedure alters the gene expression patterns and developmental potential of cloned porcine embryos

Various somatic cell nuclear transfer (SCNT) techniques for mammalian species have been developed to adjust species-specific procedures to oocyte-associated differences among species. Species-specific SCNT protocols may result in different expression levels of developmentally important genes that may affect embryonic development and pregnancy. In the present study, porcine oocytes were treated with demecolcine that facilitated enucleation with protruding genetic material. Enucleation and donor cell injection were performed either simultaneously with a single pipette (simplified one-step SCNT; SONT) or separately with different pipettes (conventional two-step SCNT; CTNT) as the control procedure. After blastocysts from both groups were cultured in vitro, the expression levels of developmentally important genes (OCT4, NANOG, EOMES, CDX2, GLUT-1, PolyA, and HSP70) were analyzed by real-time quantitative polymerase chain reaction. Both the developmental rate according to blastocyst stage as well as the expression levels CDX2, EOMES, and HSP70 were elevated with SONT compared to CTNT. The genes with elevated expression are known to influence trophectoderm formation and heat stress-induced arrest. These results showed that our SONT technique improved the development of SCNT porcine embryos, and increased the expression of genes that are important for placental formation and stress-induced arrest.

Effects of nanoscale ridge/groovepattern arrayed surface on in vitro differentiation of multi-potent pulp cells derived from human supernumerary teeth

Human dental pulp stem cells (DPSCs) are multi-potent mesenchymal stem cells that have several differentiation potentials. An understanding of the tissues that differentiate from these cells can provide insights for future regenerative therapeutics and tissue engineering strategies. The mesiodens is the most frequent form of supernumerary tooth from which DPSCs can differentiate into several lineages similar to cells from normal deciduous teeth. Recently, it has been shown that nanoscale structures can affect stem cell differentiation. In our presentstudy, we investigated the effects of a 250-nm nanoscale ridge/groove pattern array on the osteogenic and adipogenic differentiation of dental pulp cells from mesiodenscontaining human DPSCs. To this end, the expression of lineage specific markers after differentiation induction was analyzed by lineage specific staining and RT-PCR. The nanoscale pattern arrayed surface showed apositive effect on the adipogenic differentiation of DPSCs. There was no difference between nanoscale pattern arrayed surface and conventional surface groups onosteogenic differentiation. In conclusion, the nanoscale ridge/groove pattern arrayed surface can be used to enhance the adipogenic differentiation of DPSCs derived from mesiodens. This finding provides an improved understanding of the effects of topography on cell differentiation as well as the potential use of supernumerary tooth in regenerative dental medicine.

Modification of Pluripotency and Neural Crest-Related Genes' expression in Murine Skin-Derived Precursor Cells by Leukemia Inhibitory Factor (LIF)

Skin-derived precursor cells (SKPs) are multipotent, sphere-forming and embryonic neural crest-related precursor cells that can be isolated from dermis. It is known that the properties of porcine SKPs can be enhanced by leukemia inhibitory factor (LIF) which is an essential factor for the generation of embryonic stem cells in mice. In our present study, to enhance or maintain the properties of murine SKPs, LIF was added to the culture medium. SKPs were treated with 1,000 IU LIF for 72 hours after passage 3. Quantitative real time RT-PCR was then performed to quantify the expression of the pluripotent stem cell specific genes Oct4, Nanog, Klf4 and c-Myc, and the neural crest specific genes Snai2 and Ngfr. The results show that the expression of Oct4 is increased in murine SKPs by LIF treatment whereas the level of Ngfr is decreased under these conditions. Interestingly, LIF treatment reduced Nanog expression which is also important for cell proliferation in adult stem cells and for osteogenic induction in mesenchymal stem cells. These findings implicate LIF in the maintenance of stemness in SKPs through the suppression of lineage differentiation and in part through the control of cell proliferation.

Effect of Valproic acid, a Histone Deacetylase Inhibitor, on the Expression of Pluripotency and Neural Crest Specific Marker Genes in Murine Multipotent Skin Precursor Cells

Cells that have endogenous multipotent properties can be used as a starting source for the generation of induced pluripotent cells (iPSC). In addition, small molecules associated with epigenetic reprogramming are also widely used to enhance the multi- or pluripotency of such cells. Skin-derived precursor cells (SKPs) are multipotent, sphere-forming and embryonic neural crest-related precursor cells. These cells can be isolated from a juvenile or adult mammalian dermis. SKPs are also an efficient starting cell source for reprogramming and the generation of iPSCs because of the high expression levels of Sox2 and Klf4 in these cells as well as their endogenous multipotency. In this study, valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, was tested in the generation of iPSCs as a potential enhancer of the reprogramming potential of SKPs. SKPs were isolated from the back skins of 5-6 week old C57BL/6 X DBA/2 F1 mice. After passage 3, the SKPs was treated with 2 mM of VPA and the quantitative real time RT-PCR was performed to quantify the expression of Oct4 and Klf4 (pluripotency specific genes), and Snai2 and Ngfr (neural crest specific genes). The results show that Oct4 and Klf4 expression was decreased by VPA treatment. However, there were no significant changes in neural crest specific gene expression following VPA treatment. Hence, although VPA is one of the most potent of the HDAC inhibitors, it does not enhance the reprogramming of multipotent skin precursor cells in mice.

Effects of Knockout Serum Replacement in the Culture Medium on the Proliferation of Porcine Fetal Fibroblasts In Vitro

Human fibroblasts that maintain the structural integrity of connective tissues by secreting precursors of the extracellular matrix are typically cultured with serum. However, there are potential disadvantages of the use of serum including unnatural interactions between the cells and the potential for exposure to animal pathogens. To prevent the possible influence of serum on fibroblast cultures, we devised a serum-free growth method and present in vitro data that demonstrate its suitability for growing porcine fetal fibroblasts. These cells were grown under four different culture conditions: no serum (negative control), 10% fetal bovine serum (FBS, positive control), 10% knockout serum replacement (KSR) and 20% KSR in the medium. The proliferation rates and viabilities of the cells were investigated by counting the number of cells and trypan blue staining, respectively. The 10% FBS group showed the largest increase in the total number of cells (1.09 x 10(5) cells/ml). In terms of the rate of viable cells, the results from the KSR supplementation groups (20% KSR: 64.7%; 10% KSR: 80.6%) were similar to those from the 10% FBS group (68.5%). Moreover, supplementation with either 10% (3.0 x 10(4) cells/ml) or 20% KSR (4.8 x 10(4) cells/ml) produced similar cell growth rates. In conclusion, although KSR supplementation produces a lower cell proliferation rate than FBS, this growth condition is more effective for obtaining an appropriate number of viable porcine fetal fibroblasts in culture. Using KSR in fibroblast culture medium is thus a viable alternative to FBS.