Use of C57BL/6N mice on the variety of immunological researches

Hyun Keun Song,Dae Youn Hwang 한국실험동물학회 2017 Laboratory Animal Research Vol.33 No.2

Inbred mice are an essential animal strain for research as they can improve the reproducibility and reliability of study results. The establishment of new inbred lines is continuing, and new inbred lines are being used in many research fields. C57BL/6 is a mouse laboratory animal that has been developed and used as an inbred strain since early stage of mouse strain development, and, in the 1950s, C57BL/6 was separated into substrains by the Jackson Laboratory (C57BL/6J) and the National Institutes of Health (C57BL/6N). C57BL/6 mice have been used in immunology and antitumor activity studies since the early strain development stage. After the mouse genome was fully described, C57BL/6 mice use in many areas of research has expanded. In particular, immunological characteristics such as those related to cellmediated immunity and NK cell activity are relatively higher in C57BL/6 mice than in other mice. The C57BL/6NKorl is a stock of C57BL/6N established as part of a localization of experimental animal strategy of the Korean Food and Drug Administration. Based on analysis of single nucleotide polymorphisms (SNPs), C57BL/6NKorl is considered a genetically distinct inbred stock from other C57BL/6N. Various research efforts have been made to describe the characteristics and increase knowledge of the characteristics of C57BL/6Nkorl. The results obtained through these efforts are expected to increase the utilization of C57BL/6Nkorl as a domestic laboratory animal resource and to enhance the reliability of mouse based studies.

Comparison of responsiveness to cancer development and anti-cancer drug in three different C57BL/6N stocks

강미주,김지은,박지원,최현준,배수지,김길수,정영석,조준용,황대연,송현근 한국실험동물학회 2019 Laboratory Animal Research Vol.35 No.3

In our efforts to understand the systemic features of tumors, the importance of animal models is increasing due to the recent growth in the development of immunotherapy and targeted therapies. This has resulted in increased attention towards tumor animal models using C57BL/6N, which are mainly used in immunological studies. In this study, the C57BL/6NKorl stock and two other commercial stocks (C57BL/6NA and C57BL/N6B) are evaluated by comparing the occurrence of tumors using the syngeneic model; furthermore, we compare the response to anti-cancer drugs in the syngeneic model by evaluating survival, growth of tumors, proliferation and molecular biology analysis. In the syngeneic model using LLC (Lewis lung carcinoma) cells, the survival of mice and growth of the tumor showed a better response in the C57BL/6NKorl stock, and was dependent on the cell concentration of the dosing tumor, as compared to the other C57BL/6N stocks. However, the Ki-67 staining showed only little difference in cell proliferation within the tumor tissue each mouse stocks. Comparing the sensitivity to anti-cancer drug by examining changes in growth, volume and weight revealed that cisplatin treatment for tumor-bearing C57BL/6NKorl was more dependent on concentration. The Ki-67 staining, however, showed no difference among the C57BL/6N stocks after cisplatin treatment. The expressions of p27 and p53 tumor suppressor proteins, caspase-3 and Bax showed dose-dependent increase after exposure to cisplatin, whereas the expression of Bcl-2 was reduced in a dose-dependent manner. Furthermore, the expressions of MMP-2 and VEGF involved in metastasis, as well as inflammatory genes IL-1β, IL-6 and IL-10, showed dose-dependent decrease in tumor tissue after cisplatin exposure. Differences observed among the C57BL/6N stocks were not significant. Taken together, our studies reveal that C57BL/6NKorl has the potential of being a useful biological resource established in Korea, as it does not differ from the two commercially available C57BL/6N stocks when considering response to tumor generation and sensitivity to anti-cancer drugs using the syngeneic tumor model.

Compensatory role of C3 convertase on the strain difference for C3 protein expression in FVB/N, C3H/HeN and C57BL/6N mice

박지원,김지은,강미주,최현준,배수지,황대연 한국실험동물학회 2020 Laboratory Animal Research Vol.36 No.1

To investigate the role of complement C3 (C3) convertase on the strain difference for C3 protein expression in three inbred mice strains, we compared the levels of C2, C3 and C4 mRNA, as well as C3 protein and C3 convertase activity in the serum and liver tissue of FVB/N, C3H/HeN and C57BL/6N mice. The level of mRNA, inactive form (InACF) and active form (ACF) for C3 showed a regular pattern, which they were higher in the FVB/N and C57BL/6N mice than C3H/HeN mice. However, the level of C3b fragments (C3bα and β) derived from C3 protein were constantly maintained in the liver of FVB/N, C3H/HeN and C57BL/6N mice in spite of the strain difference on the transcriptional and translation level of C3. Especially, a reverse pattern of the level of mRNA, InACF and ACF for C3 was observed on the activity level of C3 convertase activity. The highest level of C3 convertase activity was measured in C3H/HeN mice, followed by C57BL/6N and FVB/N mice. In case of C3 convertase components, the level of C2 mRNA was higher in C3H/HeN mice than FVB/N and C57BL/6 N mice, while levels of C4 mRNA were higher in FVB/N and C57BL/6N mice than C3H/HeN mice. The current study results provide the first scientific evidence that C3 convertase may play complementary role to overcome the strain difference on the C3 protein expression in FVB/N, C3H/HeN and C57BL/6N mice.

Comparison of humoral and cell-mediated immunity in three different C57BL/6N mouse substrains

Hye Ryeong Kim,Jun Young Choi,Kil Soo Kim,Young-Suk Jung,Joon Young Cho,Dae Youn Hwang,Hyun Keun Song 한국실험동물학회 2017 Laboratory Animal Research Vol.33 No.2

Adaptive immunity is a type of immune response mediated by T and B cells, and is important response for immune response amplification and memory. In this study, the adaptive immunologic properties of C57BL/6NKorl substrain were compared with those of two other C57BL/6N substrains. There were no significant differences between the C57BL/6NKorl and the two other C57BL/6N substrains in the histological structures of the thymus and spleen, which are immunologic organs containing T cell and B cells. In addition, flow cytometric analysis did not reveal any significant differences in the distribution of T and B cell populations of the three substrains. To evaluate cell-mediated immunity of T cells in the three different substrains, we treated isolated T cells from spleen with Con A. The T cells of C57BL/6NKorl showed Con A-dependent proliferation of T cells at lower cell number than those in T cells from the other two C57BL/6N substrains. B cell-mediated humoral immune responses were not significant different among the three substrains. Thus, the results of this study provide evidence that C57BL/6NKorl mice are similar to those two other C57BL/6N substrains in humoral immunity, but C57BL/6NKorl has stronger response in cell mediated immunity.

Compensatory role of C3 convertase on the strain difference for C3 protein expression in FVB/N, C3H/HeN and C57BL/6N mice

Ji Won Park 한국실험동물학회 2021 한국실험동물학회 학술발표대회 논문집 Vol.2021 No.7

To investigate the role of complement C3 (C3) convertase on the strain difference for C3 protein expression in three inbred mice strains, we compared the levels of C2, C3 and C4 mRNA, as well as C3 protein and C3 convertase activity in the serum and liver tissue of FVB/N, C3H/HeN and C57BL/6N mice. The level of mRNA, inactive form (InACF) and active form (ACF) for C3 showed a regular pattern, which they were higher in the FVB/N and C57BL/6N mice than C3H/HeN mice. However, the level of C3b fragments (C3bα and β) derived from C3 protein were constantly maintained in the liver of FVB/N, C3H/HeN and C57BL/6N mice in spite of the strain difference on the transcriptional and translation level of C3. Especially, a reverse pattern of the level of mRNA, InACF and ACF for C3 was observed on the activity level of C3 convertase activity. The highest level of C3 convertase activity was measured in C3H/HeN mice, followed by C57BL/6N and FVB/N mice. In case of C3 convertase components, the level of C2 mRNA was higher in C3H/HeN mice than FVB/N and C57BL/6 N mice, while levels of C4 mRNA were higher in FVB/N and C57BL/6N mice than C3H/HeN mice. The current study results provide the first scientific evidence that C3 convertase may play complementary role to overcome the strain difference on the C3 protein expression in FVB/N, C3H/HeN and C57BL/6N mice.

Comparative study of the immunological characteristics of three different C57BL/6N mouse substrains

Jun Young Choi,Hye Ryeong Kim,Kil Soo Kim,Young-Suk Jung,Joon Young Cho,Dae Youn Hwang,Hyun Keun Song 한국실험동물학회 2017 Laboratory Animal Research Vol.33 No.2

Inbred mice, a systematically developed homogeneous animal, have been developed to maintain experimental reproducibility and to minimize experimental variables in animal-based studies. In particular, C57BL/6 mice are frequently used in experiments into immunology and antitumor activity experiments. This study was compared the immunological characteristics of C57BL/6NKorl, a Korean developed experimental animal resource, with those of two other C57BL/6N substrains. Mouse body, thymus, and spleen weights in C57BL/6NKorl were not significantly different from those of the other two C57BL/6N substrains. Among the three substrains, there was no difference in the distribution of T and B cells, which are lymphocytes involved in adaptive immunity, and no difference in NK cells related to innate immunity. Results for macrophages and granulocytes, which have roles in innate immunity, were similar in all three substrains. In order to investigate the expressions of major histocompatibility complex (MHC) molecules and allogenic antigens, splenocytes were separated from obtained spleen and analyzed by using flow cytometry. MHC class I and II molecules, which are important during self/non-self-discrimination, were similar in the three substrains. In addition, expression of alloantigen involved in allografts showed similar results in the three substrain. Thus, the results of this study provide strong evidence that C57BL/6NKorl is immunologically similar to two other C57BL/6N substrains.

Comparative Studies on Behavioral, Cognitive and Biomolecular Profiling of ICR, C57BL/6 and Its Sub-Strains Suitable for Scopolamine-Induced Amnesic Models

Karthivashan, Govindarajan,Park, Shin-Young,Kim, Joon-Soo,Cho, Duk-Yeon,Ganesan, Palanivel,Choi, Dong-Kug MDPI 2017 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.18 No.8

<P>Cognitive impairment and behavioral disparities are the distinctive baseline features to investigate in most animal models of neurodegenerative disease. However, neuronal complications are multifactorial and demand a suitable animal model to investigate their underlying basal mechanisms. By contrast, the numerous existing neurodegenerative studies have utilized various animal strains, leading to factual disparity. Choosing an optimal mouse strain for preliminary assessment of neuronal complications is therefore imperative. In this study, we systematically compared the behavioral, cognitive, cholinergic, and inflammatory impairments of outbred ICR and inbred C57BL/6 mice strains subject to scopolamine-induced amnesia. We then extended this study to the sub-strains C57BL/6N and C57BL/6J, where in addition to the above-mentioned parameters, their endogenous antioxidant levels and cAMP response-element binding protein (CREB)/brain-derived neurotrophic factor (BDNF) protein expression were also evaluated. Compared with the ICR strain, the scopolamine-inflicted C57BL/6 strains exhibited a substantial reduction of spontaneous alternation and an approximately two-fold increase in inflammatory protein expression, compared to the control group. Among the sub-strains, scopolamine-treated C57BL/6N strains exhibited declined step-through latency, elevated acetylcholinesterase (AChE) activity and inflammatory protein expression, associated with reduced endogenous antioxidant levels and p-CREB/BDNF expression, compared to the control and tacrine-treated groups. This indicates that the C57BL/6N strains exhibit significantly enhanced scopolamine-induced neuronal impairment compared to the other evaluated strains.</P>

Higher incidence of sperm granuloma in the epididymis of C57BL/6N mice

Do-Yong Park,Byoung-Seok Lee,Woo-Jin Kim,Wan-Jung Im,Ji-Seok Han,Heejin Park,Mi-Jin Yang,Jae-Woo Cho,Da Hee Kim,Su-Cheol Han,Yong-Bum Kim,Hwa-Young Son 한국실험동물학회 2018 Laboratory Animal Research Vol.34 No.1

C57BL/6N mice are inbred strains widely used in biomedical research. Hence, a large amount of basic data has been accumulated. However, in the field of histopathology, spontaneous data for relatively younger mice that are used more frequently are not yet abundant, in contrast to data for older mice and their neoplastic lesions. To acquire the essential background data required by various research and toxicological assessments, 120 mice of the C57BL/6N strain (10 and 13 weeks of age) were collected from two institutions (From Korea and Japan) and subjected to histopathological analyses of the major organs (liver, spleen, kidney, thymus, heart, testis, epididymis). The results showed significantly higher incidence of sperm granulomas in the epididymides (10-56%) of these mice, compared with that in other strains or species of lab animals. Upon closer inspection, oligospermia/clear cell hyperplasia, cellular debris, and tubular vacuolation were also observed in the epididymides with sperm granulomas. Moreover, diseased organs were significantly heavier than healthy ones. Immunohistochemical staining showed a significant increase in the chromatic figures of cysteine-dependent aspartate-directed proteases-3 (caspase-3) and cleaved-poly(ADP-ribose) polymerase (c-PARP), and damages to the tubule due to spontaneous apoptosis, which may have led to the sperms leaking out of the tubule, causing the granuloma. To conclude, spontaneous sperm granuloma can occur in 10- and 13-week-old C57BL/6N mice and may thus affect the results of various studies using these mice. Therefore, sperm granuloma in epididymis needs to be carefully considered as an important factor when design the study using C57BL/6N.

Sensitivity to tumor development by TALEN-mediated Trp53 mutant genes in the susceptible FVB/N mice and the resistance C57BL/6 mice

윤우빈,김지은,이미림,최준영,박진주,송보람,강병철,남기택,이한웅,황대연 한국실험동물학회 2021 Laboratory Animal Research Vol.37 No.4

Background This study was undertaken to compare the sensitivities of mice strains during tumor induction by transcription activator-like effector nucleases (TALEN)-mediated Trp53 mutant gene. Alterations of their tumorigenic phenotypes including survival rate, tumor formation and tumor spectrum, were assessed in FVB/N-Trp53em2Hwl/Korl and C57BL/6-Trp53em1Hwl/Korl knockout (KO) mice over 16 weeks. Results Most of the physiological phenotypes factors were observed to be higher in FVB/N-Trp53em2Hwl/Korl KO mice than C57BL/6-Trp53em1Hwl/Korl KO mice, although there were significant differences in the body weight, immune organ weight, number of red blood cells, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), total bilirubin (Bil-T) and glucose (Glu) levels in the KO mice relative to the wild type (WT) mice. Furthermore, numerous solid tumors were also observed in various regions of the surface skin of FVB/N-Trp53em2Hwl/Korl KO mice, but were not detected in C57BL/6-Trp53em1Hwl/Korl KO mice. The most frequently observed tumor in both the Trp53 KO mice was malignant lymphoma, while soft tissue teratomas and hemangiosarcomas were only detected in the FVB/N-Trp53em2Hwl/Korl KO mice. Conclusions Our results indicate that the spectrum and incidence of tumors induced by the TALEN-mediated Trp53 mutant gene is greater in FVB/N-Trp53em2Hwl/Korl KO mice than C57BL/6-Trp53em1Hwl/Korl KO mice over 16 weeks.

Effect of N-ethyl-N-nitrosourea on the fatality, recovery of fertile period and breeding record in C57BL/6J mice

변용섭,남기환,김혜림,김형친,최경철 충북대학교 동물의학연구소 2013 Journal of Biomedical and Translational Research Vol.14 No.2

N-ethyl-N-nitrosourea (ENU) is a potent mutagen in a mouse model by inducing point mutation in a random manner and, in particular, causing heritable base substitutions in spermatogonia. In this study, systematic development of phenotype-driven mutant mice with large scale was carried out by using ENU. Nine-week-old male mice of C57BL/6J received intraperitoneal injection at three times with 100 mg/kg of ENU at weekly intervals for three weeks. After injections with ENU, the changes of body weight, fatality, recovery of fertile period, and breeding record were measured in these mice. Body weight lost as a result of ENU treatments was reversed after the last ENU injection. Live fertile male mice recovered from infertility from 104 to 165 days after ENU treatments were mated with C57BL/6J female mice for generation of G1 offspring. An average birth rate was 5.9 mice from 1 pair of paternal and maternal mice. All of 231 G1 offspring mice were analyzed by modified-SHIRPA with standard procedure at nine weeks of age. Among G1 mice, 166 mice were identified as mutagenic phenotypes in 20 test items. The changes in mutagenic phenotypes after ENU treatments, for instance, pattern in the region with a different color, touch escape, changes in head morphology, pupil, and teeth, and negative geotaxis etc., were found in these mice. Taken together, these results indicate that ENU may be a trans-generational mutagen in C57BL/6J mice.