المؤلفون: Satsuki Takashima, Eiichi Okamura, Yusuke Ichiyama, Kiyoto Nishi, Akio Shimizu, Chisato Watanabe, Masanaga Muto, Shoma Matsumoto, Setsuko Tsukiyama-Fujii, Tomoyuki Tsukiyama, Hisakazu Ogita, Eiichiro Nishi, Masahito Ohji, Fumihiro Sugiyama, Satoru Takahashi, Seiya Mizuno, Ken-ichi Mizutani, Masatsugu Ema

المصدر: Experimental Animals. 2024, 73(1):93-100

المؤلفون: Bat-Erdene Jargalsaikhan, Masanaga Muto, Youngeun Been, Shoma Matsumoto, Eiichi Okamura, Tadanobu Takahashi, Yutaka Narimichi, Yuuki Kurebayashi, Hideyuki Takeuchi, Takashi Shinohara, Ryo Yamamoto, Masatsugu Ema

المصدر: Viruses, Vol 16, Iss 6, p 827 (2024)

مصطلحات موضوعية: lentiviral vector, gene therapy, Sendai virus, hemagglutinin-neuraminidase, sialic acid, hematopoietic stem cells, Microbiology, QR1-502

الوصف: A gene delivery system utilizing lentiviral vectors (LVs) requires high transduction efficiency for successful application in human gene therapy. Pseudotyping allows viral tropism to be expanded, widening the usage of LVs. While vesicular stomatitis virus G (VSV-G) single-pseudotyped LVs are commonly used, dual-pseudotyping is less frequently employed because of its increased complexity. In this study, we examined the potential of phenotypically mixed heterologous dual-pseudotyped LVs with VSV-G and Sendai virus hemagglutinin-neuraminidase (SeV-HN) glycoproteins, termed V/HN-LV. Our findings demonstrated the significantly improved transduction efficiency of V/HN-LV in various cell lines of mice, cynomolgus monkeys, and humans compared with LV pseudotyped with VSV-G alone. Notably, V/HN-LV showed higher transduction efficiency in human cells, including hematopoietic stem cells. The efficient incorporation of wild-type SeV-HN into V/HN-LV depended on VSV-G. SeV-HN removed sialic acid from VSV-G, and the desialylation of VSV-G increased V/HN-LV infectivity. Furthermore, V/HN-LV acquired the ability to recognize sialic acid, particularly N-acetylneuraminic acid on the host cell, enhancing LV infectivity. Overall, VSV-G and SeV-HN synergistically improve LV transduction efficiency and broaden its tropism, indicating their potential use in gene delivery.

وصف الملف: electronic resource

العلاقة: https://www.mdpi.com/1999-4915/16/6/827Test; https://doaj.org/toc/1999-4915Test

الوصول الحر: https://doaj.org/article/255f316f770a4b36b37e3597637ec09bTest

المؤلفون: Ayato ENDO, Daigo UCHINO, Eiichi OKAMURA, Hideaki KATO, Jumpei KURODA, Kazuki OGAWA, Keigo IKEDA, Takayoshi NARITA, Taro KATO

المصدر: Journal of the Japan Society of Applied Electromagnetics and Mechanics. 2023, 31(2):172

المؤلفون: Shoma Matsumoto, Eiichi Okamura, Masanaga Muto, Masatsugu Ema

المصدر: Reproductive Medicine and Biology, Vol 22, Iss 1, Pp n/a-n/a (2023)

مصطلحات موضوعية: Macaca fascicularis, placenta, primates, stem cells, trophoblasts, Diseases of the endocrine glands. Clinical endocrinology, RC648-665, Reproduction, QH471-489

الوصف: Abstract Background The placenta is an extraembryonic organ, which is essential to maintain a normal pregnancy. However, placental development in humans is poorly understood because of technical and ethical reasons. Methods We analyzed the anatomical localization of each trophoblastic subtype in the cynomolgus monkey placenta by immunohistochemistry in the early second trimester. Histological differences among the mouse, cynomolgus monkey, and human placenta were compared. The PubMed database was used to search for studies on placentation in rodents and primates. Main findings The anatomical structures and subtypes of the placenta in cynomolgus monkeys are highly similar to those in humans, with the exception of fewer interstitial extravillous trophoblasts in cynomolgus monkeys. Conclusion The cynomolgus monkey appears to be a good animal model to investigate human placentation.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/1445-5781Test; https://doaj.org/toc/1447-0578Test

الوصول الحر: https://doaj.org/article/366fef3e74724d589aab3ad45aff0d4bTest

المؤلفون: Hitomi Matsuzaki, Daichi Kuramochi, Eiichi Okamura, Katsuhiko Hirakawa, Aki Ushiki, Keiji Tanimoto

المصدر: Epigenetics & Chromatin, Vol 13, Iss 1, Pp 1-19 (2020)

مصطلحات موضوعية: Genetics, QH426-470

الوصف: Abstract Background Paternal allele-specific DNA methylation of the H19 imprinting control region (ICR) regulates imprinted expression of the Igf2/H19 genes. The molecular mechanism by which differential methylation of the H19 ICR is established during gametogenesis and maintained after fertilization, however, is not fully understood. We previously showed that a 2.9-kb H19 ICR fragment in transgenic mice was differentially methylated only after fertilization, demonstrating that two separable events, gametic and post-fertilization methylation, occur at the H19 ICR. We then determined that CTCF/Sox-Oct motifs and the 478-bp sequence of the H19 ICR are essential for maintaining its maternal hypomethylation status and for acquisition of paternal methylation, respectively, during the post-fertilization period. Results Using a series of 5′-truncated H19 ICR transgenes to dissect the 478-bp sequence, we identified a 118-bp region required for post-fertilization methylation activity. Deletion of the sequence from the paternal endogenous H19 ICR caused loss of methylation after fertilization, indicating that methylation activity of the sequence is required to protect endogenous H19 ICR from genome-wide reprogramming. We then reconstructed a synthetic DNA fragment in which the CTCF binding sites, Sox-Oct motifs, as well as the 118-bp sequence, were inserted into lambda DNA, and used it to replace the endogenous H19 ICR. The fragment was methylated during spermatogenesis; moreover, its allele-specific methylation status was faithfully maintained after fertilization, and imprinted expression of the both Igf2 and H19 genes was recapitulated. Conclusions Our results identified a 118-bp region within the H19 ICR that is required for de novo DNA methylation of the paternally inherited H19 ICR during pre-implantation period. A lambda DNA-based artificial fragment that contains the 118-bp sequence, in addition to the previously identified cis elements, could fully replace the function of the H19 ICR in the mouse genome.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/1756-8935Test

الوصول الحر: https://doaj.org/article/1f7ea56af0ad4c87b62e39f2e2a06ed1Test

المؤلفون: Chisato Watanabe, Hirotoshi Shibuya, Yusuke Ichiyama, Eiichi Okamura, Setsuko Tsukiyama-Fujii, Tomoyuki Tsukiyama, Shoma Matsumoto, Jun Matsushita, Takuya Azami, Yoshiaki Kubota, Masahito Ohji, Fumihiro Sugiyama, Satoru Takahashi, Seiya Mizuno, Masaru Tamura, Ken-ichi Mizutani, Masatsugu Ema

المصدر: Life, Vol 12, Iss 11, p 1730 (2022)

مصطلحات موضوعية: exocyst complex, exocyst complex component 3, cardiovascular development, hemorrhage, knock-out mouse, Exoc3l2, Science

الوصف: Angiogenesis is a process to generate new blood vessels from pre-existing vessels and to maintain vessels, and plays critical roles in normal development and disease. However, the molecular mechanisms underlying angiogenesis are not fully understood. This study examined the roles of exocyst complex component (Exoc) 3-like 2 (Exoc3l2) during development in mice. We found that Exoc3l1, Exoc3l2, Exoc3l3 and Exoc3l4 are expressed abundantly in endothelial cells at embryonic day 8.5. The generation of Exoc3l2 knock-out (KO) mice showed that disruption of Exoc3l2 resulted in lethal in utero. Substantial numbers of Exoc3l2 KO embryos exhibited hemorrhaging. Deletion of Exoc3l2 using Tie2-Cre transgenic mice demonstrated that Exoc3l2 in hematopoietic and endothelial lineages was responsible for the phenotype. Taken together, these findings reveal that Exoc3l2 is essential for cardiovascular and brain development in mice.

وصف الملف: electronic resource

العلاقة: https://www.mdpi.com/2075-1729/12/11/1730Test; https://doaj.org/toc/2075-1729Test

الوصول الحر: https://doaj.org/article/16101a5a24c24dcf90a746ede01f262bTest

المؤلفون: Hitomi Matsuzaki, Eiichi Okamura, Daichi Kuramochi, Aki Ushiki, Katsuhiko Hirakawa, Akiyoshi Fukamizu, Keiji Tanimoto

المصدر: Epigenetics & Chromatin, Vol 11, Iss 1, Pp 1-11 (2018)

مصطلحات موضوعية: Genomic imprinting, DNA methylation, H19, CTCF, Sox-Oct, ZFP57, Genetics, QH426-470

الوصف: Abstract Background Genomic imprinting is governed by allele-specific DNA methylation at imprinting control regions (ICRs), and the mechanism controlling its differential methylation establishment during gametogenesis has been a subject of intensive research interest. However, recent studies have reported that gamete methylation is not restricted at the ICRs, thus highlighting the significance of ICR methylation maintenance during the preimplantation period where genome-wide epigenetic reprogramming takes place. Using transgenic mice (TgM), we previously demonstrated that the H19 ICR possesses autonomous activity to acquire paternal-allele-specific DNA methylation after fertilization. Furthermore, this activity is indispensable for the maintenance of imprinted methylation at the endogenous H19 ICR during the preimplantation period. In addition, we showed that a specific 5′ fragment of the H19 ICR is required for its paternal methylation after fertilization, while CTCF and Sox-Oct motifs are essential for its maternal protection from undesirable methylation after implantation. Results To ask whether specific cis elements are sufficient to reconstitute imprinted methylation status, we employed a TgM co-placement strategy for facilitating detection of postfertilization methylation activity and precise comparison of test sequences. Bacteriophage lambda DNA becomes highly methylated regardless of its parental origin and thus can be used as a neutral sequence bearing no inclination for differential DNA methylation. We previously showed that insertion of only CTCF and Sox-Oct binding motifs from the H19 ICR into a lambda DNA (LCb) decreased its methylation level after both paternal and maternal transmission. We therefore appended a 478-bp 5′ sequence from the H19 ICR into the LCb fragment and found that it acquired paternal-allele-specific methylation, the dynamics of which was identical to that of the H19 ICR, in TgM. Crucially, transgene expression also became imprinted. Although there are potential binding sites for ZFP57 (a candidate protein thought to control the methylation imprint) in the larger H19 ICR, they are not found in the 478-bp fragment, rendering the role of ZFP57 in postfertilization H19 ICR methylation a still open question. Conclusions Our results demonstrate that a differentially methylated region can be reconstituted by combining the activities of specific imprinting elements and that these elements together determine the activity of a genomically imprinted region in vivo.

وصف الملف: electronic resource

العلاقة: http://link.springer.com/article/10.1186/s13072-018-0207-zTest; https://doaj.org/toc/1756-8935Test

الوصول الحر: https://doaj.org/article/85ecc0464d084e6a908773e21c6d3b7dTest

المؤلفون: Keiji Tanimoto, Hitomi Matsuzaki, Eiichi Okamura, Aki Ushiki, Akiyoshi Fukamizu, James Douglas Engel

المصدر: PLoS ONE, Vol 14, Iss 2, p e0203099 (2019)

مصطلحات موضوعية: Medicine, Science

الوصف: Long-range associations between enhancers and their target gene promoters have been shown to play critical roles in executing genome function. Recent variations of chromosome capture technology have revealed a comprehensive view of intra- and interchromosomal contacts between specific genomic sites. The locus control region of the β-globin genes (β-LCR) is a super-enhancer that is capable of activating all of the β-like globin genes within the locus in cis through physical interaction by forming DNA loops. CTCF helps to mediate loop formation between LCR-HS5 and 3'HS1 in the human β-globin locus, in this way thought to contribute to the formation of a "chromatin hub". The β-globin locus is also in close physical proximity to other erythrocyte-specific genes located long distances away on the same chromosome. In this case, erythrocyte-specific genes gather together at a shared "transcription factory" for co-transcription. Theoretically, enhancers could also activate target gene promoters at the identical loci, yet on different chromosomes in trans, a phenomenon originally described as transvection in Drosophilla. Although close physical proximity has been reported for the β-LCR and the β-like globin genes when integrated at the mouse homologous loci in trans, their structural and functional interactions were found to be rare, possibly because of a lack of suitable regulatory elements that might facilitate such trans interactions. Therefore, we re-evaluated presumptive transvection-like enhancer-promoter communication by introducing CTCF binding sites and erythrocyte-specific transcription units into both LCR-enhancer and β-promoter alleles, each inserted into the mouse ROSA26 locus on separate chromosomes. Following cross-mating of mice to place the two mutant loci at the identical chromosomal position and into active chromation in trans, their transcriptional output was evaluated. The results demonstrate that there was no significant functional association between the LCR and the β-globin gene in trans even in this idealized experimental context.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/1932-6203Test

الوصول الحر: https://doaj.org/article/9a5766e22ffc4a61942913a675c6d3f2Test

المؤلفون: Eiichi Okamura, Hitomi Matsuzaki, Akiyoshi Fukamizu, Keiji Tanimoto

المصدر: PLoS ONE, Vol 8, Iss 9, p e73925 (2013)

مصطلحات موضوعية: Medicine, Science

الوصف: Mono-allelic expression at the mouse IGF2/H19 locus is controlled by differential allelic DNA methylation of the imprinting control region (ICR). Because a randomly integrated H19 ICR fragment, when incorporated into the genome of transgenic mice (TgM), was allele-specifically methylated in somatic, but not in germ cells, it was suggested that allele-discriminating epigenetic signature, set within or somewhere outside of the Tg H19 ICR fragment in germ cells, was later translated into a differential DNA methylation pattern. To test if the chicken β-globin HS4 (cHS4) chromatin insulator might interfere with methylation imprinting establishment at the H19 ICR, we inserted the H19 ICR fragment, flanked by a set of floxed cHS4 core sequences, into a human β-globin locus YAC and generated TgM (insulated ICR' TgM). As controls, the cHS4 sequences were removed from one side (5'HS4-deleted ICR') or both sides (pseudo-WT ICR') of the insulated ICR' by in vivo cre-loxP recombination. The data show that while maternally inherited transgenic H19 ICR was not methylated in insulated ICR' TgM, it was significantly methylated upon paternal transmission, though the level was lower than in the pseudo-WT ICR' control. Because this reduced level of methylation was also observed in the 5'HS4-deleted ICR' TgM, we speculate that the phenotype is due to VEZF1-dependent demethylation activity, rather than the insulator function, borne in cHS4. Collectively, although we cannot rule out the possibility that cHS4 is incapable of blocking an allele-discriminating signal from outside of the transgene, the epigenetic signature appears to be marked intrinsically within the H19 ICR.

وصف الملف: electronic resource

العلاقة: http://europepmc.org/articles/PMC3762768?pdf=renderTest; https://doaj.org/toc/1932-6203Test

الوصول الحر: https://doaj.org/article/742b643a90f34627a123bcd73374fdb8Test

المؤلفون: Akio SHIMIZU, Chisato WATANABE, Eiichi OKAMURA, Eiichiro NISHI, Fumihiro SUGIYAMA, Hisakazu OGITA, Ken-ichi MIZUTANI, Kiyoto NISHI, Masahito OHJI, Masanaga MUTO, Masatsugu EMA, Satoru TAKAHASHI, Satsuki TAKASHIMA, Seiya MIZUNO, Setsuko TSUKIYAMA-FUJII, Shoma MATSUMOTO, Tomoyuki TSUKIYAMA, Yusuke ICHIYAMA

المصدر: Experimental Animals. :23-0105