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3. Targeting retrovirus-derived Rtl8a and 8b causes late onset obesity and neurodevelopmental defects

Author

Fujioka, Yoshifumi, primary, Shiura, Hirosuke, additional, Ishii, Masayuki, additional, Ono, Ryuichi, additional, Endo, Tsutomu, additional, Kiyonari, Hiroshi, additional, Hirate, Yoshikazu, additional, Ito, Hikaru, additional, Kanai-Azuma, Masami, additional, Kohda, Takashi, additional, Kaneko-Ishino, Tomoko, additional, and Ishino, Fumitoshi, additional

Published
2023
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13. PEG10 viral aspartic protease domain is essential for the maintenance of fetal capillary structure in the mouse placenta

Author

Shiura, Hirosuke, Ono, Ryuichi, Tachibana, Saori, Kohda, Takashi, Kaneko-Ishino, Tomoko, and Ishino, Fumitoshi

Subjects
Research Report, Reproductive Biology, Eutherian placenta evolution, viruses, Placental vascular dysfunction, Placenta, Amino Acid Motifs, Endothelial Cells, RNA-Binding Proteins, Rtl1, Pregnancy Proteins, Peg10, Capillaries, DNA-Binding Proteins, Mice, Pregnancy, embryonic structures, Placenta development, Animals, Female, Endothelium, Vascular, Gene domestication (exaptation), Apoptosis Regulatory Proteins
Abstract

The therian-specific gene paternally expressed 10 (Peg10) plays an essential role in placenta formation: Peg10 knockout mice exhibit early embryonic lethality as a result of severe placental defects. The PEG10 protein exhibits homology with long terminal repeat (LTR) retrotransposon GAG and POL proteins; therefore, we generated mice harboring a mutation in the highly conserved viral aspartic protease motif in the POL-like region of PEG10 because this motif is essential for the life cycle of LTR retrotransposons/retroviruses. Intriguingly, frequent perinatal lethality, not early embryonic lethality, was observed with fetal and placental growth retardation starting mid-gestation. In the mutant placentas, severe defects were observed in the fetal vasculature, where PEG10 is expressed in the three trophoblast cell layers that surround fetal capillary endothelial cells. Thus, Peg10 has essential roles, not only in early placenta formation, but also in placental vasculature maintenance from mid- to late-gestation. This implies that along the feto-maternal placenta interface an interaction occurs between two retrovirus-derived genes, Peg10 and retrotransposon Gag like 1 (Rtl1, also called Peg11), that is essential for the maintenance of fetal capillary endothelial cells., Summary: Disruption of the highly conserved viral aspartic protease domain in PEG10 causes placental abnormality leading to perinatal lethality in mice.

Published
2021

16. Immunoglobulin A–specific deficiency induces spontaneous inflammation specifically in the ileum

Author

Nagaishi, Takashi, primary, Watabe, Taro, additional, Kotake, Kunihiko, additional, Kumazawa, Toshihiko, additional, Aida, Tomomi, additional, Tanaka, Kohichi, additional, Ono, Ryuichi, additional, Ishino, Fumitoshi, additional, Usami, Takako, additional, Miura, Takamasa, additional, Hirakata, Satomi, additional, Kawasaki, Hiroko, additional, Tsugawa, Naoya, additional, Yamada, Daiki, additional, Hirayama, Kazuhiro, additional, Yoshikawa, Soichiro, additional, Karasuyama, Hajime, additional, Okamoto, Ryuichi, additional, Watanabe, Mamoru, additional, Blumberg, Richard S, additional, and Adachi, Takahiro, additional

Published
2021
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26. Immunoglobulin A–specific deficiency induces spontaneous inflammation specifically in the ileum

Author

Nagaishi, Takashi, Watabe, Taro, Kotake, Kunihiko, Kumazawa, Toshihiko, Aida, Tomomi, Tanaka, Kohichi, Ono, Ryuichi, Ishino, Fumitoshi, Usami, Takako, Miura, Takamasa, Hirakata, Satomi, Kawasaki, Hiroko, Tsugawa, Naoya, Yamada, Daiki, Hirayama, Kazuhiro, Yoshikawa, Soichiro, Karasuyama, Hajime, Okamoto, Ryuichi, Watanabe, Mamoru, Blumberg, Richard S, and Adachi, Takahiro

Abstract

ObjectiveAlthough immunoglobulin A (IgA) is abundantly expressed in the gut and known to be an important component of mucosal barriers against luminal pathogens, its precise function remains unclear. Therefore, we tried to elucidate the effect of IgA on gut homeostasis maintenance and its mechanism.DesignWe generated various IgA mutant mouse lines using the CRISPR/Cas9 genome editing system. Then, we evaluated the effect on the small intestinal homeostasis, pathology, intestinal microbiota, cytokine production, and immune cell activation using intravital imaging.ResultsWe obtained two lines, with one that contained a <50 base pair deletion in the cytoplasmic region of the IgA allele (IgA tail-mutant; IgAtm/tm) and the other that lacked the most constant region of the IgH α chain, which resulted in the deficiency of IgA production (IgA−/−). IgA−/−exhibited spontaneous inflammation in the ileum but not the other parts of the gastrointestinal tract. Associated with this, there were significantly increased lamina propria CD4+T cells, elevated productions of IFN-γ and IL-17, increased ileal segmented filamentous bacteria and skewed intestinal microflora composition. Intravital imaging using Ca2+biosensor showed that IgA−/−had elevated Ca2+signalling in Peyer’s patch B cells. On the other hand, IgAtm/tmseemed to be normal, suggesting that the IgA cytoplasmic tail is dispensable for the prevention of the intestinal disorder.ConclusionIgA plays an important role in the mucosal homeostasis associated with the regulation of intestinal microbiota and protection against mucosal inflammation especially in the ileum.

Published
2022
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30. Identification of a large novel imprinted gene cluster on mouse proximal chromosome 6

Author

Ono, Ryuichi, Shiura, Hirosuke, Aburatani, Hiroyuki, Kohda, Takashi, Kaneko-Ishino, Tomoko, and Ishino, Fumitoshi

Subjects
Reproduction -- Genetic aspects, Genetic regulation -- Analysis, Gene expression -- Physiological aspects, Chromosomes -- Genetic aspects, Genomes -- Research, Genetic research -- Analysis, Health
Abstract

Research has been conducted on duplication of proximal chromosome 6. The authors report the analysis of paternally expressed and maternally expressed imprinted genes within 1-Mb region, and demonstrate the existence of a large imprinted gene cluster in this region.

Published
2003

31. Double strand break repair by capture of retrotransposon sequences and reverse-transcribed spliced mRNA sequences in mouse zygotes

Author

1000010401358, Ono, Ryuichi, Ishii, Masayuki, 1000070578848, Fujihara, Yoshitaka, 1000040801965, Kitazawa, Moe, Usami, Takako, 1000020221757, Kaneko, Tomoko, 1000090186172, Kanno, Jun, 1000020304066, Ikawa, Masahito, 1000060159754, Ishino, Fumitoshi, 1000010401358, Ono, Ryuichi, Ishii, Masayuki, 1000070578848, Fujihara, Yoshitaka, 1000040801965, Kitazawa, Moe, Usami, Takako, 1000020221757, Kaneko, Tomoko, 1000090186172, Kanno, Jun, 1000020304066, Ikawa, Masahito, 1000060159754, and Ishino, Fumitoshi

Abstract

Ono, R., Ishii, M., Fujihara, Y. et al. Double strand break repair by capture of retrotransposon sequences and reverse-transcribed spliced mRNA sequences in mouse zygotes. Sci Rep 5, 12281 (2015). https://doi.org/10.1038/srep12281, The CRISPR/Cas system efficiently introduces double strand breaks (DSBs) at a genomic locus specified by a single guide RNA (sgRNA). The DSBs are subsequently repaired through non-homologous end joining (NHEJ) or homologous recombination (HR). Here, we demonstrate that DSBs introduced into mouse zygotes by the CRISPR/Cas system are repaired by the capture of DNA sequences deriving from retrotransposons, genomic DNA, mRNA and sgRNA. Among 93 mice analysed, 57 carried mutant alleles and 22 of them had long de novo insertion(s) at DSB-introduced sites; two were spliced mRNAs of Pcnt and Inadl without introns, indicating the involvement of reverse transcription (RT). Fifteen alleles included retrotransposons, mRNAs, and other sequences without evidence of RT. Two others were sgRNAs with one containing T7 promoter-derived sequence suggestive of a PCR product as its origin. In conclusion, RT-product-mediated DSB repair (RMDR) and non-RMDR repair were identified in the mouse zygote. We also confirmed that both RMDR and non-RMDR take place in CRISPR/Cas transfected NIH-3T3 cells. Finally, as two de novo MuERV-L insertions in C57BL/6 mice were shown to have characteristic features of RMDR in natural conditions, we hypothesize that RMDR contributes to the emergence of novel DNA sequences in the course of evolution.

Published
2015

34. Sirh7/Ldoc1 knockout mice exhibit placental P4 overproduction and delayed parturition

Author

Naruse, Mie, primary, Ono, Ryuichi, additional, Irie, Masahito, additional, Nakamura, Kenji, additional, Furuse, Tamio, additional, Hino, Toshiaki, additional, Oda, Kanako, additional, Kashimura, Misho, additional, Yamada, Ikuko, additional, Wakana, Shigeharu, additional, Yokoyama, Minesuke, additional, Ishino, Fumitoshi, additional, and Kaneko-Ishino, Tomoko, additional

Published
2014
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35. In Vivo Function and Evolution of the Eutherian-Specific Pluripotency Marker UTF1

Author

Nishimoto, Masazumi, primary, Katano, Miyuki, additional, Yamagishi, Toshiyuki, additional, Hishida, Tomoaki, additional, Kamon, Masayoshi, additional, Suzuki, Ayumu, additional, Hirasaki, Masataka, additional, Nabeshima, Yoko, additional, Nabeshima, Yo-ichi, additional, Katsura, Yukako, additional, Satta, Yoko, additional, Deakin, Janine E., additional, Graves, Jennifer A. Marshall, additional, Kuroki, Yoko, additional, Ono, Ryuichi, additional, Ishino, Fumitoshi, additional, Ema, Masatsugu, additional, Takahashi, Satoru, additional, Kato, Hidemasa, additional, and Okuda, Akihiko, additional

Published
2013
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36. Retrotransposon silencing by DNA methylation can drive mammalian genomic imprinting

Author

Suzuki, Shunsuke, Ono, Ryuichi, Narita, Takanori, Pask, Andrew J, Shaw, Geoffrey, Wang, Changshan, Kohda, Takashi, Alsop, Amber, Graves, Jennifer, Kohara, Yuji, Ishino, Fumitoshi, Renfree, Marilyn B, Kaneko-Ishino, Tomoko, Suzuki, Shunsuke, Ono, Ryuichi, Narita, Takanori, Pask, Andrew J, Shaw, Geoffrey, Wang, Changshan, Kohda, Takashi, Alsop, Amber, Graves, Jennifer, Kohara, Yuji, Ishino, Fumitoshi, Renfree, Marilyn B, and Kaneko-Ishino, Tomoko

Abstract

Among mammals, only eutherians and marsupials are viviparous and have genomic imprinting that leads to parent-of-origin-specific differential gene expression. We used comparative analysis to investigate the origin of genomic imprinting in mammals. PEG10 (paternally expressed 10) is a retrotransposon-derived imprinted gene that has an essential role for the formation of the placenta of the mouse. Here, we show that an orthologue of PEG10 exists in another therian mammal, the marsupial tammar wallaby (Macropus eugenii), but not in a prototherian mammal, the egg-laying platypus (Ornithorhynchus anatinus), suggesting its close relationship to the origin of placentation in therian mammals. We have discovered a hitherto missing link of the imprinting mechanism between eutherians and marsupials because tammar PEG10 is the first example of a differentially methylated region (DMR) associated with genomic imprinting in marsupials. Surprisingly, the marsupial DMR was strictly limited to the 5′ region of PEG10, unlike the eutherian DMR, which covers the promoter regions of both PEG10 and the adjacent imprinted gene SGCE. These results not only demonstrate a common origin of the DMR-associated imprinting mechanism in therian mammals but provide the first demonstration that DMR-associated genomic imprinting in eutherians can originate from the repression of exogenous DNA sequences and/or retrotransposons by DNA methylation.

Published
2007

37. Role of retrotransposon-derived imprinted gene, Rtl1, in the feto-maternal interface of mouse placenta

Author

Sekita, Yoichi, primary, Wagatsuma, Hirotaka, additional, Nakamura, Kenji, additional, Ono, Ryuichi, additional, Kagami, Masayo, additional, Wakisaka, Noriko, additional, Hino, Toshiaki, additional, Suzuki-Migishima, Rika, additional, Kohda, Takashi, additional, Ogura, Atsuo, additional, Ogata, Tsutomu, additional, Yokoyama, Minesuke, additional, Kaneko-Ishino, Tomoko, additional, and Ishino, Fumitoshi, additional

Published
2008
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38. Retrotransposon Silencing by DNA Methylation Can Drive Mammalian Genomic Imprinting

Author

Suzuki, Shunsuke, primary, Ono, Ryuichi, additional, Narita, Takanori, additional, Pask, Andrew J, additional, Shaw, Geoffrey, additional, Wang, Changshan, additional, Kohda, Takashi, additional, Alsop, Amber E, additional, Marshall Graves, Jennifer A., additional, Kohara, Yuji, additional, Ishino, Fumitoshi, additional, Renfree, Marilyn B, additional, and Kaneko-Ishino, Tomoko, additional

Published
2007
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39. Deletion of Peg10, an imprinted gene acquired from a retrotransposon, causes early embryonic lethality

Author

Ono, Ryuichi, primary, Nakamura, Kenji, additional, Inoue, Kimiko, additional, Naruse, Mie, additional, Usami, Takako, additional, Wakisaka-Saito, Noriko, additional, Hino, Toshiaki, additional, Suzuki-Migishima, Rika, additional, Ogonuki, Narumi, additional, Miki, Hiromi, additional, Kohda, Takashi, additional, Ogura, Atsuo, additional, Yokoyama, Minesuke, additional, Kaneko-Ishino, Tomoko, additional, and Ishino, Fumitoshi, additional

Published
2005
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40. Erratum to “characterization and imprinting status of OBPH1/Obph1 gene: implications for an extended imprinting domain in human and mouse”

Author

Higashimoto, Ken, primary, Soejima, Hidenobu, additional, Yatsuki, Hitomi, additional, Joh, Keiichiro, additional, Uchiyama, Michiko, additional, Obata, Yayoi, additional, Ono, Ryuichi, additional, Wang, Youdong, additional, Xin, Zhenghan, additional, Zhu, Xike, additional, Masuko, Sadahiko, additional, Ishino, Fumitoshi, additional, Hatada, Izuho, additional, Jinno, Yoshihiro, additional, Iwasaka, Tsuyoshi, additional, Katsuki, Takeshi, additional, and Mukai, Tsunehiro, additional

Published
2003
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42. MousePeg9/Dlk1and humanPEG9/DLK1are paternally expressed imprinted genes closely located to the maternally expressed imprinted genes: mouseMeg3/Gtl2and humanMEG3

Author

Kobayashi, Shin, primary, Wagatsuma, Hirotaka, additional, Ono, Ryuichi, additional, Ichikawa, Hitoshi, additional, Yamazaki, Masaaki, additional, Tashiro, Hiroyuki, additional, Aisaka, Kohzo, additional, Miyoshi, Naoki, additional, Kohda, Takashi, additional, Ogura, Atsuo, additional, Ohki, Misao, additional, Kaneko-Ishino, Tomoko, additional, and Ishino, Fumitoshi, additional

Published
2000
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43. Identification of a Novel PNMA-MS1 Gene in Marsupials Suggests the LTR Retrotransposon-Derived PNMA Genes Evolved Differently in Marsupials and Eutherians.

Author

Iwasaki, Sawa, Suzuki, Shunsuke, Pelekanos, Matthew, Clark, Helen, Ono, Ryuichi, Shaw, Geoff, Renfree, Marilyn B., Kaneko-Ishino, Tomoko, and Ishino, Fumitoshi

Abstract

Two major gene families derived from Ty3/Gypsy long terminal repeat (LTR) retrotransposons were recently identified in mammals. The sushi-ichi retrotransposon homologue (SIRH) family comprises 12 genes: 11 in eutherians including Peg10 and Peg11/Rtl1 that have essential roles in the eutherian placenta and 1 that is marsupial specific. Fifteen and 12 genes were reported in the second gene family, para-neoplastic antigen MA (PNMA), in humans and mice, respectively, although their biological functions and evolutionary history remain largely unknown. Here, we identified two novel candidate PNMA genes, PNMA-MS1 and -MS2 in marsupials. Like all eutherian-specific PNMA genes, they exhibit the highest homology to a Gypsy12_DR (DR, Danio rerio) Gag protein. PNMA-MS1 is conserved in both Australian and South American marsupial species, the tammar wallaby and grey short-tailed opossum. However, no PNMA-MS1 orthologue was found in eutherians, monotremes or non-mammalian vertebrates. PNMA-MS1 was expressed in the ovary, mammary gland and brain during development and growth in the tammar, suggesting that PNMA-MS1 may have acquired a marsupial-specific function. However, PNMA-MS2 seems to be a pseudogene. The absence of marsupial orthologues of eutherian PNMA genes suggests that the retrotransposition events of the Gypsy12_DR-related retrotransposons that gave rise to the PNMA family occurred after the divergence of marsupials and eutherians. [ABSTRACT FROM PUBLISHER]

Published
2013
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44. In Vivo Function and Evolution of the Eutherian-Specific Pluripotency Marker UTF1.

Author

Nishimoto, Masazumi, Katano, Miyuki, Yamagishi, Toshiyuki, Hishida, Tomoaki, Kamon, Masayoshi, Suzuki, Ayumu, Hirasaki, Masataka, Nabeshima, Yoko, Nabeshima, Yo-ichi, Katsura, Yukako, Satta, Yoko, Deakin, Janine E., Graves, Jennifer A. Marshall, Kuroki, Yoko, Ono, Ryuichi, Ishino, Fumitoshi, Ema, Masatsugu, Takahashi, Satoru, Kato, Hidemasa, and Okuda, Akihiko

Subjects
BIOMARKERS, MAMMAL embryology, GENE expression in mammals, EMBRYONIC stem cells, GENETIC engineering, DEVELOPMENTAL biology, BIOLOGICAL evolution
Abstract

Embryogenesis in placental mammals is sustained by exquisite interplay between the embryo proper and placenta. UTF1 is a developmentally regulated gene expressed in both cell lineages. Here, we analyzed the consequence of loss of the UTF1 gene during mouse development. We found that homozygous UTF1 mutant newborn mice were significantly smaller than wild-type or heterozygous mutant mice, suggesting that placental insufficiency caused by the loss of UTF1 expression in extra-embryonic ectodermal cells at least in part contributed to this phenotype. We also found that the effects of loss of UTF1 expression in embryonic stem cells on their pluripotency were very subtle. Genome structure and sequence comparisons revealed that the UTF1 gene exists only in placental mammals. Our analyses of a family of genes with homology to UTF1 revealed a possible mechanism by which placental mammals have evolved the UTF1 genes. [ABSTRACT FROM AUTHOR]

Published
2013
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45. Identification of tammar wallaby SIRH12, derived from a marsupial-specific retrotransposition event.

Author

Ono, Ryuichi, Kuroki, Yoko, Naruse, Mie, Ishii, Masayuki, Iwasaki, Sawa, Toyoda, Atsushi, Fujiyama, Asao, Shaw, Geoff, Renfree, Marilyn B., Kaneko-Ishino, Tomoko, and Ishino, Fumitoshi

Abstract

In humans and mice, there are 11 genes derived from sushi-ichi related retrotransposons, some of which are known to play essential roles in placental development. Interestingly, this family of retrotransposons was thought to exist only in eutherian mammals, indicating their significant contributions to the eutherian evolution, but at least one, PEG10, is conserved between marsupials and eutherians. Here we report a novel sushi-ichi retrotransposon-derived gene, SIRH12, in the tammar wallaby, an Australian marsupial species of the kangaroo family. SIRH12 encodes a protein highly homologous to the sushi-ichi retrotransposon Gag protein in the tammar wallaby, while SIRH12 in the South American short-tailed grey opossum is a pseudogene degenerated by accumulation of multiple nonsense mutations. This suggests that SIRH12 retrotransposition occurred only in the marsupial lineage but acquired and retained some as yet unidentified novel function, at least in the lineage of the tammar wallaby. [ABSTRACT FROM PUBLISHER]

Published
2011
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46. Mouse Peg9/Dlk1 and humanPEG9/DLK1 are paternally expressed imprinted genes closely located to the maternally expressed imprinted genes: mouseMeg3/Gtl2 and humanMEG3.

Author

Kobayashi, Shin, Wagatsuma, Hirotaka, Ono, Ryuichi, Ichikawa, Hitoshi, Yamazaki, Masaaki, Tashiro, Hiroyuki, Aisaka, Kohzo, Miyoshi, Naoki, Kohda, Takashi, Ogura, Atsuo, Ohki, Misao, Kaneko-Ishino, Tomoko, and Ishino, Fumitoshi

Subjects
GENOMIC imprinting, GENE expression
Abstract

Genomic imprinting significantly influences development, growth and behaviour in mammals. Systematic screening of imprinted genes has been extensively carried out to identify the genes responsible for imprinted phenotypes and to elucidate the biological significance of this phenomenon. In this study, we applied DNA chip technology for isolating paternally expressed imprinted genes (Pegs). We compared the resulting expression profiles of parthenogenetic and fertilized control embryos to identify novel imprinted genes. A novel paternally expressed mouse imprinted gene, Peg9/Dlk1, was identified. Consistent with this finding, the paternal expression of its human homologue, PEG9/DLK1, was also confirmed. These two genes form imprinted gene clusters with the reciprocally imprinted mouse Meg3/Gtl2 and human MEG3 genes that we first identified on distal chromosome 12 and chromosome 14q32, respectively. As DNA chip technology allows us to quickly screen a large number of genes, using this technology to search for imprinted genes could accelerate the identification of genes responsible for human and mouse genetic diseases. Dlk1 and DLK1, which encode transmembrane proteins, have six EGF-like repeats and show homology to the Delta gene in Drosophila melanogaster. Because of its homology to mammalian Delta homologues, PEG9/DLK1 may contribute to the scoliosis phenotype observed in maternal uniparental disomy 14 (mUPD14) patients. [ABSTRACT FROM AUTHOR]

Published
2000
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47. A Retrotransposon-Derived Gene, PEG10,Is a Novel Imprinted Gene Located on Human Chromosome 7q21

Author

Ono, Ryuichi, Kobayashi, Shin, Wagatsuma, Hirotaka, Aisaka, Kohzo, Kohda, Takashi, Kaneko-Ishino, Tomoko, and Ishino, Fumitoshi

Abstract

A novel paternally expressed imprinted gene, PEG10(Paternally Expressed 10), was identified on human chromosome 7q21. PEG10is located near the SGCE(Sarcoglycan ϵ) gene, whose mouse homologue was recently shown to be imprinted. Therefore, it is highly possible that a new imprinted gene cluster exists on human chromosome 7q21. Analysis of two predicted open reading frames (ORF1 and ORF2) revealed that ORF1 and ORF2 have homology to the gag and pol proteins of some vertebrate retrotransposons, respectively. These data suggest that PEG10is derived from a retrotransposon that was previously integrated into the mammalian genome. PEG10is likely to be essential for understanding how exogenous genes become imprinted.

Published
2001
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48. Double strand break repair by capture of retrotransposon sequences and reverse-transcribed spliced mRNA sequences in mouse zygotes

Author

Ono, Ryuichi, Ishii, Masayuki, Fujihara, Yoshitaka, Kitazawa, Moe, Usami, Takako, Kaneko, Tomoko, Kanno, Jun, Ikawa, Masahito, Ishino, Fumitoshi, Ono, Ryuichi, Ishii, Masayuki, Fujihara, Yoshitaka, Kitazawa, Moe, Usami, Takako, Kaneko, Tomoko, Kanno, Jun, Ikawa, Masahito, and Ishino, Fumitoshi

Abstract

Ono, R., Ishii, M., Fujihara, Y. et al. Double strand break repair by capture of retrotransposon sequences and reverse-transcribed spliced mRNA sequences in mouse zygotes. Sci Rep 5, 12281 (2015). https://doi.org/10.1038/srep12281, The CRISPR/Cas system efficiently introduces double strand breaks (DSBs) at a genomic locus specified by a single guide RNA (sgRNA). The DSBs are subsequently repaired through non-homologous end joining (NHEJ) or homologous recombination (HR). Here, we demonstrate that DSBs introduced into mouse zygotes by the CRISPR/Cas system are repaired by the capture of DNA sequences deriving from retrotransposons, genomic DNA, mRNA and sgRNA. Among 93 mice analysed, 57 carried mutant alleles and 22 of them had long de novo insertion(s) at DSB-introduced sites; two were spliced mRNAs of Pcnt and Inadl without introns, indicating the involvement of reverse transcription (RT). Fifteen alleles included retrotransposons, mRNAs, and other sequences without evidence of RT. Two others were sgRNAs with one containing T7 promoter-derived sequence suggestive of a PCR product as its origin. In conclusion, RT-product-mediated DSB repair (RMDR) and non-RMDR repair were identified in the mouse zygote. We also confirmed that both RMDR and non-RMDR take place in CRISPR/Cas transfected NIH-3T3 cells. Finally, as two de novo MuERV-L insertions in C57BL/6 mice were shown to have characteristic features of RMDR in natural conditions, we hypothesize that RMDR contributes to the emergence of novel DNA sequences in the course of evolution.

49. Double strand break repair by capture of retrotransposon sequences and reverse-transcribed spliced mRNA sequences in mouse zygotes

Author

Ono, Ryuichi, Ishii, Masayuki, Fujihara, Yoshitaka, Kitazawa, Moe, Usami, Takako, Kaneko, Tomoko, Kanno, Jun, Ikawa, Masahito, Ishino, Fumitoshi, Ono, Ryuichi, Ishii, Masayuki, Fujihara, Yoshitaka, Kitazawa, Moe, Usami, Takako, Kaneko, Tomoko, Kanno, Jun, Ikawa, Masahito, and Ishino, Fumitoshi

Abstract

Ono, R., Ishii, M., Fujihara, Y. et al. Double strand break repair by capture of retrotransposon sequences and reverse-transcribed spliced mRNA sequences in mouse zygotes. Sci Rep 5, 12281 (2015). https://doi.org/10.1038/srep12281, The CRISPR/Cas system efficiently introduces double strand breaks (DSBs) at a genomic locus specified by a single guide RNA (sgRNA). The DSBs are subsequently repaired through non-homologous end joining (NHEJ) or homologous recombination (HR). Here, we demonstrate that DSBs introduced into mouse zygotes by the CRISPR/Cas system are repaired by the capture of DNA sequences deriving from retrotransposons, genomic DNA, mRNA and sgRNA. Among 93 mice analysed, 57 carried mutant alleles and 22 of them had long de novo insertion(s) at DSB-introduced sites; two were spliced mRNAs of Pcnt and Inadl without introns, indicating the involvement of reverse transcription (RT). Fifteen alleles included retrotransposons, mRNAs, and other sequences without evidence of RT. Two others were sgRNAs with one containing T7 promoter-derived sequence suggestive of a PCR product as its origin. In conclusion, RT-product-mediated DSB repair (RMDR) and non-RMDR repair were identified in the mouse zygote. We also confirmed that both RMDR and non-RMDR take place in CRISPR/Cas transfected NIH-3T3 cells. Finally, as two de novo MuERV-L insertions in C57BL/6 mice were shown to have characteristic features of RMDR in natural conditions, we hypothesize that RMDR contributes to the emergence of novel DNA sequences in the course of evolution.

50. [Percellome Project: research on molecular mechanisms of toxicological responses based on transcriptomics and epigenetics].

Author

Aisaki KI, Ono R, Kanno J, and Kitajima S

Subjects
Animals, Epigenesis, Genetic, Genomics, Mice, Toxicogenetics methods, Artificial Intelligence, Transcriptome
Abstract

We are constructing the "Percellome Database" containing many transcriptomes of mice exposed to a series of chemicals to elucidate the molecular mechanism of toxicity and to develop toxicity prediction technology. Acute toxicity of a chemical can be predicted to a certain extent by searching the similarity of the transcriptomes obtained by the single-dose exposure experiments. In addition, we are analyzing the relation between the transcriptome and the epigenome i.e. histone modification and genomic DNA methylation to understand the molecular mechanism of the repeated dose toxicity. We are attempting to expand the scale and improve the efficiency of the analysis by introducing artificial intelligence technologies. This approach should maximize the use of toxicogenomics technology for optimizing the experimental protocols for repeated dose toxicity studies towards 3Rs principle, and optimizing the process of in silico toxicity prediction by combining the available big data.

Published
2022
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