Reproductive engineering techniques are essential for assisted reproduction of animals and generation of genetically modified animals. They may also provide invaluable research models for understanding the mechanisms involved in the developmental and reproductive processes. At the RIKEN BioResource Center (BRC), I have sought to develop new reproductive engineering techniques, especially those related to cryopreservation, microinsemination (sperm injection), nuclear transfer, and generation of new stem cell lines and animals, hoping that they will support the present and future projects at BRC. I also want to combine our techniques with genetic and biochemical analyses to solve important biological questions. We expect that this strategy makes our research more unique and refined by providing deeper insights into the mechanisms that govern the reproductive and developmental systems in mammals. To make this strategy more effective, it is critical to work with experts in different scientific fields. I have enjoyed collaborations with about 100 world-recognized laboratories, and all our collaborations have been successful and fruitful. This review summarizes development of reproductive engineering techniques at BRC during these 15 years.
{"title":"Development of reproductive engineering techniques at the RIKEN BioResource Center","authors":"A. Ogura","doi":"10.1538/expanim.16-0074","DOIUrl":"https://doi.org/10.1538/expanim.16-0074","url":null,"abstract":"Reproductive engineering techniques are essential for assisted reproduction of animals and generation of genetically modified animals. They may also provide invaluable research models for understanding the mechanisms involved in the developmental and reproductive processes. At the RIKEN BioResource Center (BRC), I have sought to develop new reproductive engineering techniques, especially those related to cryopreservation, microinsemination (sperm injection), nuclear transfer, and generation of new stem cell lines and animals, hoping that they will support the present and future projects at BRC. I also want to combine our techniques with genetic and biochemical analyses to solve important biological questions. We expect that this strategy makes our research more unique and refined by providing deeper insights into the mechanisms that govern the reproductive and developmental systems in mammals. To make this strategy more effective, it is critical to work with experts in different scientific fields. I have enjoyed collaborations with about 100 world-recognized laboratories, and all our collaborations have been successful and fruitful. This review summarizes development of reproductive engineering techniques at BRC during these 15 years.","PeriodicalId":75961,"journal":{"name":"Jikken dobutsu. Experimental animals","volume":"66 1","pages":"1 - 16"},"PeriodicalIF":0.0,"publicationDate":"2016-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1538/expanim.16-0074","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"67291200","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Minoru Katō, Yi‐Ying Huang, Mina Matsuo, Yoko Takashina, K. Sasaki, Yasushi Horai, Aya Juni, Shin-ichi Kamijo, K. Saigo, K. Ui-Tei, H. Tei
RNA interference (RNAi) is a powerful tool for the study of gene function in mammalian systems, including transgenic mice. Here, we report a gene knockdown system based on the human mir-187 precursor. We introduced small interfering RNA (siRNA) sequences against the mouse melanocortin-4 receptor (mMc4r) to alter the targeting of miR-187. The siRNA-expressing cassette was placed under the control of the cytomegalovirus (CMV) early enhancer/chicken β-actin promoter. In vitro, the construct efficiently knocked down the gene expression of a co-transfected mMc4r-expression vector in cultured mammalian cells. Using this construct, we generated a transgenic mouse line which exhibited partial but significant knockdown of mMc4r mRNA in various brain regions. Northern blot analysis detected transgenic expression of mMc4r siRNA in these regions. Furthermore, the transgenic mice fed a normal diet ate 9% more and were 30% heavier than wild-type sibs. They also developed hyperinsulinemia and fatty liver as do mMc4r knockout mice. We determined that this siRNA expression construct based on mir-187 is a practical and useful tool for gene functional studies in vitro as well as in vivo.
{"title":"RNAi-mediated knockdown of mouse melanocortin-4 receptor in vitro and in vivo, using an siRNA expression construct based on the mir-187 precursor","authors":"Minoru Katō, Yi‐Ying Huang, Mina Matsuo, Yoko Takashina, K. Sasaki, Yasushi Horai, Aya Juni, Shin-ichi Kamijo, K. Saigo, K. Ui-Tei, H. Tei","doi":"10.1538/expanim.16-0065","DOIUrl":"https://doi.org/10.1538/expanim.16-0065","url":null,"abstract":"RNA interference (RNAi) is a powerful tool for the study of gene function in mammalian systems, including transgenic mice. Here, we report a gene knockdown system based on the human mir-187 precursor. We introduced small interfering RNA (siRNA) sequences against the mouse melanocortin-4 receptor (mMc4r) to alter the targeting of miR-187. The siRNA-expressing cassette was placed under the control of the cytomegalovirus (CMV) early enhancer/chicken β-actin promoter. In vitro, the construct efficiently knocked down the gene expression of a co-transfected mMc4r-expression vector in cultured mammalian cells. Using this construct, we generated a transgenic mouse line which exhibited partial but significant knockdown of mMc4r mRNA in various brain regions. Northern blot analysis detected transgenic expression of mMc4r siRNA in these regions. Furthermore, the transgenic mice fed a normal diet ate 9% more and were 30% heavier than wild-type sibs. They also developed hyperinsulinemia and fatty liver as do mMc4r knockout mice. We determined that this siRNA expression construct based on mir-187 is a practical and useful tool for gene functional studies in vitro as well as in vivo.","PeriodicalId":75961,"journal":{"name":"Jikken dobutsu. Experimental animals","volume":"66 1","pages":"41 - 50"},"PeriodicalIF":0.0,"publicationDate":"2016-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1538/expanim.16-0065","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"67291417","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
We previously showed that prepubertal chronic caffeine exposure adversely affected the development of the testes in male rats. Here we investigated dose- and time-related effects of caffeine consumption on the testis throughout sexual maturation in prepubertal rats. A total of 80 male SD rats were randomly divided into four groups: controls and rats fed 20, 60, or 120 mg caffeine/kg/day, respectively, via gavage for 10, 20, 30, or 40 days. Preputial separation was monitored daily before the rats were sacrificed. Terminal blood samples were collected for hormone assay, and testes were grossly evaluated and weighed. One testis was processed for histological analysis, and the other was collected to isolate Leydig cells. Caffeine exposure significantly increased the relative weight of the testis in a dose-related manner after 30 days of exposure, whereas the absolute testis weight tended to decrease at the 120 mg dose of caffeine. The mean diameter of the seminiferous tubules and height of the germinal epithelium significantly decreased in the caffeine-fed groups after 40 days of caffeine exposure, which was accompanied by a reduced BrdU incorporation rate in germ cells. In addition, caffeine intake significantly reduced in vivo and ex vivo testosterone production in a dose-related manner. Our results demonstrate that caffeine exposure during sexual maturation alter the testicular microarchitecture and also slow germ cell proliferation even at the 20 mg dose level. Furthermore, caffeine may act directly on Leydig cells and interfere with testosterone production in a dose-related manner, consequently delaying onset of sexual maturation.
{"title":"Dose- and time-related effects of caffeine on the testis in immature male rats","authors":"J. Bae, Hyeonhae Choi, Yuri Choi, J. Roh","doi":"10.1538/expanim.16-0060","DOIUrl":"https://doi.org/10.1538/expanim.16-0060","url":null,"abstract":"We previously showed that prepubertal chronic caffeine exposure adversely affected the development of the testes in male rats. Here we investigated dose- and time-related effects of caffeine consumption on the testis throughout sexual maturation in prepubertal rats. A total of 80 male SD rats were randomly divided into four groups: controls and rats fed 20, 60, or 120 mg caffeine/kg/day, respectively, via gavage for 10, 20, 30, or 40 days. Preputial separation was monitored daily before the rats were sacrificed. Terminal blood samples were collected for hormone assay, and testes were grossly evaluated and weighed. One testis was processed for histological analysis, and the other was collected to isolate Leydig cells. Caffeine exposure significantly increased the relative weight of the testis in a dose-related manner after 30 days of exposure, whereas the absolute testis weight tended to decrease at the 120 mg dose of caffeine. The mean diameter of the seminiferous tubules and height of the germinal epithelium significantly decreased in the caffeine-fed groups after 40 days of caffeine exposure, which was accompanied by a reduced BrdU incorporation rate in germ cells. In addition, caffeine intake significantly reduced in vivo and ex vivo testosterone production in a dose-related manner. Our results demonstrate that caffeine exposure during sexual maturation alter the testicular microarchitecture and also slow germ cell proliferation even at the 20 mg dose level. Furthermore, caffeine may act directly on Leydig cells and interfere with testosterone production in a dose-related manner, consequently delaying onset of sexual maturation.","PeriodicalId":75961,"journal":{"name":"Jikken dobutsu. Experimental animals","volume":"66 1","pages":"29 - 39"},"PeriodicalIF":0.0,"publicationDate":"2016-09-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1538/expanim.16-0060","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"67291371","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The Japan Aerospace Exploration Agency developed the mouse Habitat Cage Unit (HCU) for installation in the Cell Biology Experiment Facility (CBEF) onboard the Japanese Experimental Module ("Kibo") on the International Space Station. The CBEF provides "space-based controls" by generating artificial gravity in the HCU through a centrifuge, enabling a comparison of the biological consequences of microgravity and artificial gravity of 1 g on mice housed in space. Therefore, prior to the space experiment, a ground-based study to validate the habitability of the HCU is necessary to conduct space experiments using the HCU in the CBEF. Here, we investigated the ground-based effect of a 32-day housing period in the HCU breadboard model on male mice in comparison with the control cage mice. Morphology of skeletal muscle, the thymus, heart, and kidney, and the sperm function showed no critical abnormalities between the control mice and HCU mice. Slight but significant changes caused by the HCU itself were observed, including decreased body weight, increased weights of the thymus and gastrocnemius, reduced thickness of cortical bone of the femur, and several gene expressions from 11 tissues. Results suggest that the HCU provides acceptable conditions for mouse phenotypic analysis using CBEF in space, as long as its characteristic features are considered. Thus, the HCU is a feasible device for future space experiments.
{"title":"Ground-based assessment of JAXA mouse habitat cage unit by mouse phenotypic studies.","authors":"Miki Shimbo, Takashi Kudo, Michito Hamada, Hyojung Jeon, Yuki Imamura, Keigo Asano, Risa Okada, Yuki Tsunakawa, Seiya Mizuno, Ken-Ichi Yagami, Chihiro Ishikawa, Haiyan Li, Takashi Shiga, Junji Ishida, Juri Hamada, Kazuya Murata, Tomohiro Ishimaru, Misuzu Hashimoto, Akiyoshi Fukamizu, Mutsumi Yamane, Masahito Ikawa, Hironobu Morita, Masahiro Shinohara, Hiroshi Asahara, Taishin Akiyama, Nobuko Akiyama, Hiroki Sasanuma, Nobuaki Yoshida, Rui Zhou, Ying-Ying Wang, Taito Ito, Yuko Kokubu, Taka-Aki K Noguchi, Hisako Ishimine, Akira Kurisaki, Dai Shiba, Hiroyasu Mizuno, Masaki Shirakawa, Naoki Ito, Shin Takeda, Satoru Takahashi","doi":"10.1538/expanim.15-0077","DOIUrl":"10.1538/expanim.15-0077","url":null,"abstract":"<p><p>The Japan Aerospace Exploration Agency developed the mouse Habitat Cage Unit (HCU) for installation in the Cell Biology Experiment Facility (CBEF) onboard the Japanese Experimental Module (\"Kibo\") on the International Space Station. The CBEF provides \"space-based controls\" by generating artificial gravity in the HCU through a centrifuge, enabling a comparison of the biological consequences of microgravity and artificial gravity of 1 g on mice housed in space. Therefore, prior to the space experiment, a ground-based study to validate the habitability of the HCU is necessary to conduct space experiments using the HCU in the CBEF. Here, we investigated the ground-based effect of a 32-day housing period in the HCU breadboard model on male mice in comparison with the control cage mice. Morphology of skeletal muscle, the thymus, heart, and kidney, and the sperm function showed no critical abnormalities between the control mice and HCU mice. Slight but significant changes caused by the HCU itself were observed, including decreased body weight, increased weights of the thymus and gastrocnemius, reduced thickness of cortical bone of the femur, and several gene expressions from 11 tissues. Results suggest that the HCU provides acceptable conditions for mouse phenotypic analysis using CBEF in space, as long as its characteristic features are considered. Thus, the HCU is a feasible device for future space experiments. </p>","PeriodicalId":75961,"journal":{"name":"Jikken dobutsu. Experimental animals","volume":"65 1","pages":"175-87"},"PeriodicalIF":0.0,"publicationDate":"2016-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4873486/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"67290111","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Yoshikazu Hasegawa, Yoshikazu Hoshino, Abdelaziz E. Ibrahim, Kanako Kato, Yoko Daitoku, Y. Tanimoto, Y. Ikeda, Hisashi Oishi, Satoru Takahashi, A. Yoshiki, K. Yagami, Hiroyoshi Iseki, S. Mizuno, F. Sugiyama
In the present study, we generated novel cre driver mice for gene manipulation in pancreatic β cells. Using the CRISPR/Cas9 system, stop codon sequences of Ins1 were targeted for insertion of cre, including 2A sequences. A founder of C57BL/6J-Ins1em1 (cre) Utr strain was produced from an oocyte injected with pX330 containing the sequences encoding gRNA and Cas9 and a DNA donor plasmid carrying 2A-cre. (R26GRR x C57BL/6J-Ins1em1 (cre) Utr) F1 mice were histologically characterized for cre-loxP recombination in the embryonic and adult stages; cre-loxP recombination was observed in all pancreatic islets examined in which almost all insulin-positive cells showed tdsRed fluorescence, suggesting β cell-specific recombination. Furthermore, there were no significant differences in results of glucose tolerance test among genotypes (homo/hetero/wild). Taken together, these observations indicated that C57BL/6J-Ins1em1 (cre) Utr is useful for studies of glucose metabolism and the strategy of bicistronic cre knock-in using the CRISPR/Cas9 system could be useful for production of cre driver mice.
{"title":"Generation of CRISPR/Cas9-mediated bicistronic knock-in ins1-cre driver mice","authors":"Yoshikazu Hasegawa, Yoshikazu Hoshino, Abdelaziz E. Ibrahim, Kanako Kato, Yoko Daitoku, Y. Tanimoto, Y. Ikeda, Hisashi Oishi, Satoru Takahashi, A. Yoshiki, K. Yagami, Hiroyoshi Iseki, S. Mizuno, F. Sugiyama","doi":"10.1538/expanim.16-0016","DOIUrl":"https://doi.org/10.1538/expanim.16-0016","url":null,"abstract":"In the present study, we generated novel cre driver mice for gene manipulation in pancreatic β cells. Using the CRISPR/Cas9 system, stop codon sequences of Ins1 were targeted for insertion of cre, including 2A sequences. A founder of C57BL/6J-Ins1em1 (cre) Utr strain was produced from an oocyte injected with pX330 containing the sequences encoding gRNA and Cas9 and a DNA donor plasmid carrying 2A-cre. (R26GRR x C57BL/6J-Ins1em1 (cre) Utr) F1 mice were histologically characterized for cre-loxP recombination in the embryonic and adult stages; cre-loxP recombination was observed in all pancreatic islets examined in which almost all insulin-positive cells showed tdsRed fluorescence, suggesting β cell-specific recombination. Furthermore, there were no significant differences in results of glucose tolerance test among genotypes (homo/hetero/wild). Taken together, these observations indicated that C57BL/6J-Ins1em1 (cre) Utr is useful for studies of glucose metabolism and the strategy of bicistronic cre knock-in using the CRISPR/Cas9 system could be useful for production of cre driver mice.","PeriodicalId":75961,"journal":{"name":"Jikken dobutsu. Experimental animals","volume":"65 1","pages":"319 - 327"},"PeriodicalIF":0.0,"publicationDate":"2016-04-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1538/expanim.16-0016","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"67291161","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Chi Hai-Ying, Kiori Nagano, S. Ezzikouri, Chiho Yamaguchi, M. E. H. Kayesh, K. Rebbani, B. Kitab, Hirohumi Nakano, H. Kouji, M. Kohara, K. Tsukiyama-Kohara
Previous studies have shown that intermittent cold stress (ICS) induces depression-like behaviors in mammals. Tupaia belangeri (the tree shrew) is the only experimental animal other than the chimpanzee that has been shown to be susceptible to infection by hepatitis B and C viruses. Moreover, full genome sequence analysis has revealed strong homology between host proteins in Tupaia and in humans and other primates. Tupaia neuromodulator receptor proteins are also known to have a high degree of homology with their corresponding primate proteins. Based on these similarities, we hypothesized that induction of ICS in Tupaia would provide a useful animal model of stress responses. We exposed young adult Tupaia to ICS and observed decreases in body temperature and body weight in both female and male Tupaia, suggesting that Tupaia are an appropriate animal model for ICS studies. We further examined the efficacy of a new small-molecule compound, C737, against the effects of ICS. C737 mimics the helical structure of neuron-restrictive silencer factor (NRSF/REST), which regulates a wide range of target genes involved in neuronal function and pain modulation. Treatment with C737 significantly reduced stress-induced weight loss in female Tupaia; these effects were stronger than those elicited by the antidepressant agomelatine. These results suggest that Tupaia represents a useful non-rodent ICS model. Our data also provide new insights into the function of NRSF/REST in stress-induced depression and other disorders with epigenetic influences or those with high prevalence in women.
{"title":"Establishment of an intermittent cold stress model using Tupaia belangeri and evaluation of compound C737 targeting neuron-restrictive silencer factor","authors":"Chi Hai-Ying, Kiori Nagano, S. Ezzikouri, Chiho Yamaguchi, M. E. H. Kayesh, K. Rebbani, B. Kitab, Hirohumi Nakano, H. Kouji, M. Kohara, K. Tsukiyama-Kohara","doi":"10.1538/expanim.15-0123","DOIUrl":"https://doi.org/10.1538/expanim.15-0123","url":null,"abstract":"Previous studies have shown that intermittent cold stress (ICS) induces depression-like behaviors in mammals. Tupaia belangeri (the tree shrew) is the only experimental animal other than the chimpanzee that has been shown to be susceptible to infection by hepatitis B and C viruses. Moreover, full genome sequence analysis has revealed strong homology between host proteins in Tupaia and in humans and other primates. Tupaia neuromodulator receptor proteins are also known to have a high degree of homology with their corresponding primate proteins. Based on these similarities, we hypothesized that induction of ICS in Tupaia would provide a useful animal model of stress responses. We exposed young adult Tupaia to ICS and observed decreases in body temperature and body weight in both female and male Tupaia, suggesting that Tupaia are an appropriate animal model for ICS studies. We further examined the efficacy of a new small-molecule compound, C737, against the effects of ICS. C737 mimics the helical structure of neuron-restrictive silencer factor (NRSF/REST), which regulates a wide range of target genes involved in neuronal function and pain modulation. Treatment with C737 significantly reduced stress-induced weight loss in female Tupaia; these effects were stronger than those elicited by the antidepressant agomelatine. These results suggest that Tupaia represents a useful non-rodent ICS model. Our data also provide new insights into the function of NRSF/REST in stress-induced depression and other disorders with epigenetic influences or those with high prevalence in women.","PeriodicalId":75961,"journal":{"name":"Jikken dobutsu. Experimental animals","volume":"65 1","pages":"285 - 292"},"PeriodicalIF":0.0,"publicationDate":"2016-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1538/expanim.15-0123","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"67291227","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Y. Shirasaki, A. Matsuura, Masashi Uekusa, Yoshihiro Ito, Toshiaki Hayashi
Chlorine dioxide (ClO2) is a strong oxidant that possesses an antimicrobial activity. We demonstrated here that ClO2 gas is easily generated by mixing 3.35% sodium chlorite solution (Purogene) and 85% phosphoric acid at a 10:1 volume ratio without using an expensive machine. In a test room (87 m3), experiments were carried out using various amounts of sodium chlorite solution (0.25 ml/m3 to 20.0 ml/m3). The gas concentration increased in a sodium chlorite volume-dependent manner and reached peak values of from 0.8 ppm to 40.8 ppm at 2 h–3 h, and then gradually decreased. No differences in gas concentrations were observed between 0.1 and 2.5 m above the floor, indicating that the gas was evenly distributed. Under high-humidity (approximately 80% relative humidity), colony formation of both Staphylococcus aureus and Escherichia coli was completely inhibited by ClO2 gas exposure at 1.0 ml/m3 sodium chlorite solution (mean maximal concentration of 3.0 ppm). Exposure at 4.0 ml/m3 sodium chlorite solution (mean maximal concentration of 10.6 ppm) achieved complete inactivation of Bacillus atrophaeus spores. In contrast, without humidification, the efficacy of ClO2 gas was apparently attenuated, suggesting that the atmospheric moisture is indispensable. Delicate electronic devices (computer, camera, etc.) operated normally, even after being subjected to more than 20 times of fumigation. Considering that our method for gas generation is simple, reproducible, and highly effective at decontaminating microbes, our approach is expected to serve as an inexpensive alternative method for cleaning and disinfecting animal facilities.
{"title":"A study of the properties of chlorine dioxide gas as a fumigant","authors":"Y. Shirasaki, A. Matsuura, Masashi Uekusa, Yoshihiro Ito, Toshiaki Hayashi","doi":"10.1538/expanim.15-0092","DOIUrl":"https://doi.org/10.1538/expanim.15-0092","url":null,"abstract":"Chlorine dioxide (ClO2) is a strong oxidant that possesses an antimicrobial activity. We demonstrated here that ClO2 gas is easily generated by mixing 3.35% sodium chlorite solution (Purogene) and 85% phosphoric acid at a 10:1 volume ratio without using an expensive machine. In a test room (87 m3), experiments were carried out using various amounts of sodium chlorite solution (0.25 ml/m3 to 20.0 ml/m3). The gas concentration increased in a sodium chlorite volume-dependent manner and reached peak values of from 0.8 ppm to 40.8 ppm at 2 h–3 h, and then gradually decreased. No differences in gas concentrations were observed between 0.1 and 2.5 m above the floor, indicating that the gas was evenly distributed. Under high-humidity (approximately 80% relative humidity), colony formation of both Staphylococcus aureus and Escherichia coli was completely inhibited by ClO2 gas exposure at 1.0 ml/m3 sodium chlorite solution (mean maximal concentration of 3.0 ppm). Exposure at 4.0 ml/m3 sodium chlorite solution (mean maximal concentration of 10.6 ppm) achieved complete inactivation of Bacillus atrophaeus spores. In contrast, without humidification, the efficacy of ClO2 gas was apparently attenuated, suggesting that the atmospheric moisture is indispensable. Delicate electronic devices (computer, camera, etc.) operated normally, even after being subjected to more than 20 times of fumigation. Considering that our method for gas generation is simple, reproducible, and highly effective at decontaminating microbes, our approach is expected to serve as an inexpensive alternative method for cleaning and disinfecting animal facilities.","PeriodicalId":75961,"journal":{"name":"Jikken dobutsu. Experimental animals","volume":"65 1","pages":"303 - 310"},"PeriodicalIF":0.0,"publicationDate":"2016-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1538/expanim.15-0092","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"67290151","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ai Nishitani, Miyuu Tanaka, S. Shimizu, Naofumi Kunisawa, M. Yokoe, Yusaku Yoshida, Toshiro Suzuki, Tetsushi Sakuma, Takashi Yamamoto, M. Kuwamura, S. Takenaka, Y. Ohno, T. Kuramoto
Essential tremor (ET) is a common movement disorder with a poorly understood etiology. The TRM/Kyo mutant rat, showing spontaneous tremor, is an animal model of ET. Recently, we demonstrated that tremors in these rats emerge when two mutant loci, a missense mutation in the hyperpolarization-activated cyclic nucleotide-gated potassium channel 1 (Hcn1) and the tremor (tm) deletion, are present simultaneously. However, we did not identify which gene within the tm deletion causes tremor expression in TRM/Kyo rats. A strong candidate among the 13 genes within the tm deletion is aspartoacylase (Aspa), because some Aspa-knockout mouse strains show tremor. Here, we generated Aspa-knockout rats using transcription activator-like effector nuclease technology and produced Aspa/Hcn1 double-mutant rats by crossing Aspa-knockout rats with Hcn1-mutant rats. The Aspa-knockout rats carried nonsense mutations in exon 4; and ASPA proteins were not detectable in their brain extracts. They showed elevated levels of N-acetyl-L-aspartate (NAA) in urine and spongy vacuolation and abnormal myelination in the central nervous system, but no tremor. By contrast, Aspa/Hcn1 double-mutant rats spontaneously showed tremors resembling those in TRM/Kyo rats, and the tremor was suppressed by drugs therapeutic for ET but not for parkinsonian tremor. These findings indicated that the lack of the Aspa gene caused tremor expression in TRM/Kyo rats. Our animal model suggested that the interaction of NAA accumulation due to ASPA deficiency with an unstable neuronal membrane potential caused by HCN1 deficiency was involved in tremor development.
{"title":"Involvement of aspartoacylase in tremor expression in rats","authors":"Ai Nishitani, Miyuu Tanaka, S. Shimizu, Naofumi Kunisawa, M. Yokoe, Yusaku Yoshida, Toshiro Suzuki, Tetsushi Sakuma, Takashi Yamamoto, M. Kuwamura, S. Takenaka, Y. Ohno, T. Kuramoto","doi":"10.1538/expanim.16-0007","DOIUrl":"https://doi.org/10.1538/expanim.16-0007","url":null,"abstract":"Essential tremor (ET) is a common movement disorder with a poorly understood etiology. The TRM/Kyo mutant rat, showing spontaneous tremor, is an animal model of ET. Recently, we demonstrated that tremors in these rats emerge when two mutant loci, a missense mutation in the hyperpolarization-activated cyclic nucleotide-gated potassium channel 1 (Hcn1) and the tremor (tm) deletion, are present simultaneously. However, we did not identify which gene within the tm deletion causes tremor expression in TRM/Kyo rats. A strong candidate among the 13 genes within the tm deletion is aspartoacylase (Aspa), because some Aspa-knockout mouse strains show tremor. Here, we generated Aspa-knockout rats using transcription activator-like effector nuclease technology and produced Aspa/Hcn1 double-mutant rats by crossing Aspa-knockout rats with Hcn1-mutant rats. The Aspa-knockout rats carried nonsense mutations in exon 4; and ASPA proteins were not detectable in their brain extracts. They showed elevated levels of N-acetyl-L-aspartate (NAA) in urine and spongy vacuolation and abnormal myelination in the central nervous system, but no tremor. By contrast, Aspa/Hcn1 double-mutant rats spontaneously showed tremors resembling those in TRM/Kyo rats, and the tremor was suppressed by drugs therapeutic for ET but not for parkinsonian tremor. These findings indicated that the lack of the Aspa gene caused tremor expression in TRM/Kyo rats. Our animal model suggested that the interaction of NAA accumulation due to ASPA deficiency with an unstable neuronal membrane potential caused by HCN1 deficiency was involved in tremor development.","PeriodicalId":75961,"journal":{"name":"Jikken dobutsu. Experimental animals","volume":"65 1","pages":"293 - 301"},"PeriodicalIF":0.0,"publicationDate":"2016-03-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1538/expanim.16-0007","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"67291026","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Miho Terao, Moe Tamano, Satoshi Hara, Tomoko Kato, M. Kinoshita, S. Takada
The CRISPR/Cas9 system is a powerful genome editing tool for the production of genetically modified animals. To produce mutant mice, chimeric single-guide RNA (sgRNA) is cloned in a plasmid vector and a mixture of sgRNA and Cas9 are microinjected into the fertilized eggs. An issue associated with gene manipulation using the CRISPR/Cas9 system is that there can be off-target effects. To simplify the production of mutant mice with low risks of off-target effects caused by the CRISPR/Cas9 system, we demonstrated that genetically modified mice can be efficiently obtained using chemically synthesized CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and modified Cas9s, such as the nickase version and FokI-fused catalytically inactive Cas9, by microinjection into fertilized eggs. Using this method, it is no longer necessary to clone sgRNA into a plasmid vector, and this enables high-throughput production of mutant mice.
{"title":"Utilization of the CRISPR/Cas9 system for the efficient production of mutant mice using crRNA/tracrRNA with Cas9 nickase and FokI-dCas9","authors":"Miho Terao, Moe Tamano, Satoshi Hara, Tomoko Kato, M. Kinoshita, S. Takada","doi":"10.1538/expanim.15-0116","DOIUrl":"https://doi.org/10.1538/expanim.15-0116","url":null,"abstract":"The CRISPR/Cas9 system is a powerful genome editing tool for the production of genetically modified animals. To produce mutant mice, chimeric single-guide RNA (sgRNA) is cloned in a plasmid vector and a mixture of sgRNA and Cas9 are microinjected into the fertilized eggs. An issue associated with gene manipulation using the CRISPR/Cas9 system is that there can be off-target effects. To simplify the production of mutant mice with low risks of off-target effects caused by the CRISPR/Cas9 system, we demonstrated that genetically modified mice can be efficiently obtained using chemically synthesized CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and modified Cas9s, such as the nickase version and FokI-fused catalytically inactive Cas9, by microinjection into fertilized eggs. Using this method, it is no longer necessary to clone sgRNA into a plasmid vector, and this enables high-throughput production of mutant mice.","PeriodicalId":75961,"journal":{"name":"Jikken dobutsu. Experimental animals","volume":"65 1","pages":"275 - 283"},"PeriodicalIF":0.0,"publicationDate":"2016-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1538/expanim.15-0116","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"67290968","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Hossam H. Shawki, Takumi Kigoshi, Y. Katoh, M. Matsuda, Chioma M. Ugboma, Satoru Takahashi, Hisashi Oishi, A. Kawashima
Previously, we have identified a calcium-binding protein that is specifically expressed in spermatids and localized to the flagella of the mature sperm in mouse, so-called mCABS1. However, the physiological roles of CABS1 in the male reproductive system have not been fully elucidated yet. In the current study, we aimed to localize and clarify the role of CABS1 in porcine (pCABS1). We determined for the first time the full nucleotides sequence of pCABS1 mRNA. pCABS1 protein was detected on SDS-PAGE gel as two bands at 75 kDa and 70 kDa in adult porcine testis, whereas one band at 70 kDa in epididymal sperm. pCABS1 immunoreactivity in seminiferous tubules was detected in the elongated spermatids, and that in the epididymal sperm was found in the acrosome as well as flagellum. The immunoreactivity of pCABS1 in the acrosomai region disappeared during acrosome reaction. We also identified that pCABS1 has a transmembrane domain using computational prediction of the amino acids sequence. The treatment of porcine capacitated sperm with anti-pCABS1 antiserum significantly decreased acrosome reactions. These results suggest that pCABS1 plays an important role in controlling calcium ion signaling during the acrosome reaction.
{"title":"Identification, localization, and functional analysis of the homologues of mouse CABS1 protein in porcine testis","authors":"Hossam H. Shawki, Takumi Kigoshi, Y. Katoh, M. Matsuda, Chioma M. Ugboma, Satoru Takahashi, Hisashi Oishi, A. Kawashima","doi":"10.1538/expanim.15-0104","DOIUrl":"https://doi.org/10.1538/expanim.15-0104","url":null,"abstract":"Previously, we have identified a calcium-binding protein that is specifically expressed in spermatids and localized to the flagella of the mature sperm in mouse, so-called mCABS1. However, the physiological roles of CABS1 in the male reproductive system have not been fully elucidated yet. In the current study, we aimed to localize and clarify the role of CABS1 in porcine (pCABS1). We determined for the first time the full nucleotides sequence of pCABS1 mRNA. pCABS1 protein was detected on SDS-PAGE gel as two bands at 75 kDa and 70 kDa in adult porcine testis, whereas one band at 70 kDa in epididymal sperm. pCABS1 immunoreactivity in seminiferous tubules was detected in the elongated spermatids, and that in the epididymal sperm was found in the acrosome as well as flagellum. The immunoreactivity of pCABS1 in the acrosomai region disappeared during acrosome reaction. We also identified that pCABS1 has a transmembrane domain using computational prediction of the amino acids sequence. The treatment of porcine capacitated sperm with anti-pCABS1 antiserum significantly decreased acrosome reactions. These results suggest that pCABS1 plays an important role in controlling calcium ion signaling during the acrosome reaction.","PeriodicalId":75961,"journal":{"name":"Jikken dobutsu. Experimental animals","volume":"65 1","pages":"253 - 265"},"PeriodicalIF":0.0,"publicationDate":"2016-03-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1538/expanim.15-0104","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"67290631","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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