The 28,000-year-old remains of a woolly mammoth, named 'Yuka', were found in Siberian permafrost. Here we recovered the less-damaged nucleus-like structures from the remains and visualised their dynamics in living mouse oocytes after nuclear transfer. Proteomic analyses demonstrated the presence of nuclear components in the remains. Nucleus-like structures found in the tissue homogenate were histone- and lamin-positive by immunostaining. In the reconstructed oocytes, the mammoth nuclei showed the spindle assembly, histone incorporation and partial nuclear formation; however, the full activation of nuclei for cleavage was not confirmed. DNA damage levels, which varied among the nuclei, were comparable to those of frozen-thawed mouse sperm and were reduced in some reconstructed oocytes. Our work provides a platform to evaluate the biological activities of nuclei in extinct animal species.

Antioxidant mechanisms to adequately moderate levels of endogenous reactive oxygen species (ROS) are important for oocytes and embryos to obtain and maintain developmental competence, respectively. Immediately after fertilization, ROS levels in zygotes are elevated but the antioxidant mechanisms during the maternal-to-zygotic transition (MZT) are not well understood. First, we identified peroxiredoxin 1 (PRDX1) and PRDX2 by proteomics analysis as two of the most abundant endogenous antioxidant enzymes eliminating hydrogen peroxide (H2O2). We here report the cellular localization of hyperoxidized PRDX and its involvement in the antioxidant mechanisms of freshly fertilized oocytes. Treatment of zygotes at the pronuclear stage with H2O2 enhanced pronuclear localization of hyperoxidized PRDX in zygotes and concurrently impaired the generation of 5-hydroxymethylcytosine (5hmC) on the male genome, which is an epigenetic reprogramming event that occurs at the pronuclear stage. Thus, our results suggest that endogenous PRDX is involved in antioxidant mechanisms and epigenetic reprogramming during MZT.

The fertilized oocyte begins cleavage, leading to zygotic gene activation (ZGA), which re-activates the resting genome to acquire totipotency. In this process, genomic function is regulated by the dynamic structural conversion in the nucleus. Indeed, a considerable number of genes that are essential for embryonic development are located near the pericentromeric regions, wherein the heterochromatin is formed. These genes are repressed transcriptionally in somatic cells. Three-dimensional fluorescence in situ hybridization (3D-FISH) enables the visualization of the intranuclear spatial arrangement, such as gene loci, chromosomal domains, and chromosome territories (CTs). However, the 3D-FISH approach in mammalian embryos has been limited to certain repeated sequences because of its unfavorable properties. In this study, we developed an easy-to-use chamber device (EASI-FISH chamber) for 3D-FISH in early embryos, and visualized, for the first time, the spatial arrangements of pericentromeric regions, the ZGA-activated gene (Zscan4) loci, and CTs (chromosome 7), simultaneously during the early cleavage stage of mouse embryos by 3D-FISH. As a result, it was revealed that morphological changes of the pericentromeric regions and CTs, and relocation of the Zscan4 loci in CTs, occurred in the 1- to 4-cell stage embryos, which was different from those in somatic cells. This convenient and reproducible 3D-FISH technique for mammalian embryos represents a valuable tool that will provide insights into the nuclear dynamics of development.

C57/BL/6Jマウスを用い、β-ニコチンアミド・モノヌクレオチド(β-NMN)を体外成熟培地に用いることによる体外成熟卵子の細胞質内活性酸素種(ROS)に与える影響および体外成熟卵子の発生能に与える影響について検討した。各濃度のβ-NMNを添加したmTaM体外成熟培地による57/BL/6Jマウス由来GV期卵子を用いた体外成熟成績は、これら各濃度のβ-NMNを添加した体外成熟培地による体外成熟率は1mM区では81%(64/79)、2mM区では84%(70/83)、5mM区では83%(75/90)、10mM区では83%(69/83)で、非添加区における体外成熟後のMII期卵子発生成績74%(135/183)と比べ有意差は認められなかった。ROSの検出における蛍光輝度成熟は74%であり、非添加群と比べ、1mMおよび2mM添加区において蛍光輝度の低下を認めた。これらの結果、mTaM培地にβ-NMN添加は細胞質内のROSの過剰産生を抑制することが認められた。

[要旨] これまで遺伝資源の保存技術の構築のために, 様々な動物種の培養細胞を樹立し遺伝資源の確保と細胞特性の検討を行っている. しかし, 高度な発生工学・生殖工学的技術の習熟には時間を要し, 特に体細胞核移植操作は, 作製した再構築胚の発生率が非常に低率であることから多くの胚を作製しなければならない状況下にある. そこで本実験では, 体細胞核移植（Somatic cell nuclear transfer : SCNT）により作製した初期胚のガラス化保存を実施し, 加温後の正常性を検討した. B6D2F 1マウスへ常法に従い過剰排卵処置後にSCNT を実施した. 続いて発生した胚は簡易ガラス化法によりガラス化保存を行った. 加温後, 形態的に正常と認められた胚は, DAPI 染色および免疫染色（Oct4）による組織学的観察と胚発生を検討した. SCNT により作製し, 生存した卵子を活性化処理後, 2細胞期へ発生した再構築胚は83.2% であり, 一部は胚盤胞期へ発生した（44.8% : 47/105）. これら再構築胚をガラス化保存し一部を加温した結果, 加温後の生存胚は70% 以上であり免疫組織化学的解析により形態的な正常性を認めた. 以上の結果から, 作製されたSCNT胚のガラス化保存による遺伝資源の保存とそれらの胚を用いた移植核の内部構造構築に関する基礎的検討が可能であることが示唆された. [Abstract] Somatic cell nuclear transfer（SCNT） has been used in various research namely investigation into cellular features for establishing of genetic resourses technology from wild animals. However, it requires a high level of developmental engineering and reproduction technology which take time to learn as well as many successfully reconstructed oocytes and embryos. In this experiment, we examined the cryopreservation by simple vitrification of the mouse early embryos produced by SCNT. After warming a part of cloned embryos, morphologically normal embryos were obtained at more than 70%. And they were morphologically normal by the investigation with immunohistochemical analysis, which detected Oct4, puluripotency facter in inner cell mass. In conclusion, these results suggested that the genetic resources preservation were possible by simple vitrification of cloned mouse embryos. Furthermore, fundamental study on the internal structure of the cell nucleus can be made possible using the cloned mouse embryos.近畿大学先端技術総合研究所紀要編集委員会

In the mammalian testis, the ubiquitin-proteasome system plays important roles in the process that promotes the formation of mature sperm. We recently identified zygote-specific proteasome assembly chaperone (ZPAC), which is specifically expressed in the mouse gonads and zygote. ZPAC mediates a unique proteasome assembly pathway in the zygote, but the expression profile and function of ZPAC in the testis is not fully understood. In this study, we investigated the possible role of ZPAC during mouse spermatogenesis. First, we analyzed the expression of ZPAC and 20S proteasome subunit alpha 4/PSMA7 in the adult mouse testis. ZPAC and alpha 4 were expressed in spermatogonia, spermatocytes, and round spermatids. In elongating spermatids, ZPAC was expressed until step 10, whereas expression of alpha 4 persisted until step 12. We then examined the expression profile of ZPAC and alpha 4 in a mouse model of experimental unilateral cryptorchidism. Consistent with appearance of morphologically impaired germ cells following cryptorchidism, the ZPAC protein level was significantly decreased at 4 days post induction of experimental cryptorchidism (D4) compared with the intact testis, although the amount of alpha 4 protein persisted at least until D10. Moreover, intense ZPAC staining was co-localized with staining of annexin V, an early indicator of apoptosis in mammalian cells, in germ cells of cryptorchid testis, but ZPAC was also expressed in germ cells showing no detectable expression of annexin V. These results suggest that ZPAC plays a role during spermatogenesis and raises the possibility that 20S proteasome mediated by ZPAC may be involved in the regulation of germ cell survival during spermatogenesis.

[要旨]本実験では、野生種であるアカネズミおよびカヤネズミから少量の組織サンプルを回収し効率よく安定したDNA を回収するために、バロサイクラー（Baro Cycler）を用いてDNA 抽出とその相同性について検索を行なった。さらに、両マウスの尾部から採取した組織より線維芽細胞を樹立し、染色体解析および樹立した細胞の遺伝資源保存を検討した。その結果、尾部組織および樹立線維芽細胞それぞれはシークエンス後のBLAST 検索によってデータベース上にて高い相同性を示した。また、核型解析では、それぞれの核型は高い正常性を示した。さらに、それら体細胞を用いて、電気融合法による異属間体細胞核移植を実施したところ、一部の再構築卵子は2 細胞期胚へと発生することを認めた。以上の結果から、今回樹立した野生マウス由来線維芽細胞に異常は少なく、それら細胞の遺伝子資源の保存が可能であることが示された。 [Abstract] In this study, to improve the DNA extraction efficiencies for a small amount of tissue from two wild mice, we have done a search for DNA homology with Baro Cycler. In addition, fibroblasts were also established from those mice tails. Karyotype analyses were performed on the fibroblasts, and those cells were frozen for the preservation. As a result, the frozen-thawed fibroblast cells have a high number of normal karyotype. Fibroblasts derived from organization and tail confirmed the homology sequence after BLAST search. Furthermore, these somatic cells performed somatic cell nuclear transfer（SCNT）by electrofusion method. These results of reconstructed oocytes several developed to 2-cell stage embryos. In conclusion, abnormalities in this wild mouse derived fibroblast cells were low, and thereforeconservation of genetic resources was possible in these somatic cells.近畿大学先端技術総合研究所紀要編集委員会

Interspecies somatic cell nuclear transfer (ISCNT) has been proposed as a technique to produce cloned offspring of endangered species as well as to investigate nucleus-cytoplasm interactions in mammalian embryo. However, it is still not known which embryo culture medium is optimal for ISCNT embryos for the nuclear donor or the oocyte recipient. We assessed the effects of the culture medium on the developmental competence of the ISCNT embryos by introducing cynomolgus monkey (Macaca fascicularis) fibroblast nuclei into enucleated rabbit (Oryctolagus cuniculus) oocytes (monkey-rabbit embryo). The monkey-rabbit ISCNT embryos that were cultured in mCMRL-1066 developed to the blastocyst stage, although all monkey-rabbit ISCNT embryos cultured in M199 were arrested by the 4-cell stage. When monkey-rabbit ISCNT and rabbit-rabbit somatic cell nuclear transfer (SCNT) embryos were cultured in mCMRL-1066, the blastocyst cell numbers of the monkey-rabbit ISCNT embryos corresponded to the cell numbers of the control rabbit-rabbit SCNT embryos, which were produced from a rabbit fibroblast nucleus and an enucleated rabbit oocyte. In addition, the presence of mitochondria, which were introduced with monkey fibroblasts into rabbit recipient cytoplasm, was confirmed up to the blastocyst stage by polymerase chain reaction (PCR). This study demonstrated that: (1) rabbit oocytes can reprogramme cynomolgus monkey somatic cell nuclei, and support preimplantation development; (2) monkey-rabbit ISCNT embryos developed well in monkey culture medium at early embryonic developmental stages; (3) the cell number of monkey-rabbit ISCNT embryos is similar to that of rabbit-rabbit SCNT embryos; and (4) the mitochondrial fate of monkey-rabbit ISCNT embryos is heteroplasmic from the time just after injection to the blastocyst stage that has roots in both rabbit oocytes and monkey fibroblasts.

After fertilization, the sperm and oocyte genomes undergo extensive epigenetic reprogramming to form a totipotent zygote. The dynamic epigenetic changes during early embryo development primarily involve DNA methylation and demethylation. We have previously identified Gse (gonad-specific expression gene) to be expressed specifically in germ cells and early embryos. Its encoded protein GSE is predominantly localized in the nuclei of cells from the zygote to blastocyst stages, suggesting possible roles in the epigenetic changes occurring during early embryo development. Here, we report the involvement of GSE in epigenetic reprogramming of the paternal genome during mouse zygote development. Preferential binding of GSE to the paternal chromatin was observed from pronuclear stage 2 (PN2) onward. A knockdown of GSE by antisense RNA in oocytes produced no apparent effect on the first and second cell cycles in preimplantation embryos, but caused a significant reduction in the loss of 5-methylcytosine (5 mC) and the accumulation of 5-hydroxymethylcytosine (5 hmC) in the paternal pronucleus. Furthermore, DNA methylation levels in CpG sites of LINE1 transposable elements, Lemd1, Nanog and the upstream regulatory region of the Oct4 (also known as Pou5f1) gene were clearly increased in GSE-knockdown zygotes at mid-pronuclear stages (PN3-4), but the imprinted H19-differential methylated region was not affected. Importantly, DNA immunoprecipitation of 5 mC and 5 hmC also indicates that knockdown of GSE in zygotes resulted in a significant reduction of the conversion of 5 mC to 5 hmC on LINE1. Therefore, our results suggest an important role of maternal GSE for mediating active DNA demethylation in the zygote.

[要旨] 1994年、精原幹細胞の精細管移植によりドナー由来の精子を作製できる移植技術が開発された。さらに、2007年には幼若ブタ精巣細胞を免疫不全マウスの皮下へ移植することにより、精子形成の誘導に成功したことが報告された。そこで本研究では、絶滅危惧動物などでの精子形成分化誘導モデルの確立を目的に、ウシ精巣細胞を用いた移植実験を行った。マウス新生子（生後7日以内）、ウシ新生子（生後7 日以内）およびウシ幼若（3～5ヶ月齢）精巣細胞をマトリゲルマトリックスと混合し、免疫不全マウスの皮下に移植することで、精巣様構造の再構築と精子形成の誘導を試みた。移植後、1週～ 44週に組織学的解析を行った。さらに、生殖細胞の生存・増殖・分化について、抗VASA 抗体を用いた免疫組織化学的解析を行った。マウス新生子精巣細胞の移植では、移植後10週以降に再構築精細管および減数分裂に至る精子細胞がみられ、精子形成誘導を可能にする機能的な精細管の構築が示された。一方、新生子ならびに幼若ウシ精巣細胞の移植では、移植後10 週から管構造の再建がみられたものの、免疫組織化学的解析において再構築された精細管様構造の基底膜上に配置される生殖細胞を認めることはできなかった。以上の結果から、異種異所移植法によりウシにおいても精細管様構造が再構築されたことから, 今後移植法の改良によりウシ精子形成の誘導が可能となることが期待される。 [Abstract] Male germ cell transplantation induces donor cell-derived spermatogenesis in the testis of recipient mice. However, this technique is still available only to mice and rats. Recently, xenoectopic transplantation of testicular cells into the subcutis of immunodeficient mice has been developed as a novel technology to rebuild seminiferous tubules and to produce functional sperm. In order to develop an alternative technology for transgenesis and induction of spermatogenesis in domestic animals, we examinedxenoectopic transplantation of bovine testicular cells. Testicular cells from neonatal（within 7 days）and juvenile（3-5 months）bovine and neonatal（within 7 days）mouse testes were prepared at the concentration of 1-5 x 10^7/ml（mouse）or 1 x 10^8/ml（bovine）and then mixed with equal volume of BD Matrigel^ Basement Membrane Matrix（Matrigel）. The testicular cell/Matrigel suspensions were grafted into the subcutis of castrated immunodeficient male mice（BALB/c Slc-nu/nu）. Morphogenesis of the grafts was examined between 1 and 44 weeks after transplantation. To examine the growth and differentiation of donor germ cells, the expression of VASA, which is expressed from gonocytes to early meiotic stages in mammalian male germ cells, was examined by immunohistochemistry. Grafts derived from mouse testicular cells regenerated complete functional seminiferous tubules 20 weeks after transplantation in which haploid spermatids were generated in the lumen. Initiation of reconstruction of seminiferous tubules from bovine testicular cells was also observed 10 weeks after transplantation. However, immunohistochemical analysis demonstrated that bovine germ cells failed to colonize along the basement membrane of reconstructed seminiferous tubules and to proliferate thereafter. Although further investigation for suitable conditions of bovine germ cell colonization and survival in the reconstructed seminiferous tubules is needed, xenoectopic transplantation would make possible to induce bovine spermatogenesis and be usefull for animal sciences, conservation of wild animals and male infertility.

[要約] 本研究では、卵子の細胞内運搬経路として密接な関係を示す、L- カルニチンを添加した体外成熟培地を用いて、マウス未成熟卵子への体外成熟および体外受精をおこない、その後の初期胚の発生改善に与える影響を検討した。私たちの開発した、修正TaM 培地にL- カルニチン1mM, 2mM, 5mM, 10mM を添加して体外成熟をおこなった結果、82 ～ 90％であり、非添加区（92％：905/987）との有意な差は認めず正常に発生した。次に、体外成熟を経て正常に発生した卵子の体外受精成績は、それぞれ69 ～ 80％であり、非添加区（71％：635/889）との有意な差は認めなかった。続いて、胚盤胞期胚への発生成績は、それぞれ24 ～ 46 ％ であり、2mM添加成熟培地（46 ％：136/293） および5mM添加成熟培地（37％：101/272）では、非添加区（26％：129/499）と比較し初期胚の発生に改善を認めた。さらに、初期胚の発生が改善された、2mM L- カルニチン添加区において、正常な産子への発生を認めた。これらの結果から、体外成熟培地への適切な濃度のL- カルニチン添加は、胚盤胞期胚への発生を有意に改善することが示唆された。 [Abstract] In this study, mouse immature oocytes of ICR used in vitro maturation and in vitro fertilization, since analysised early embryos development. PMSG interperitoneal administration mouse collected immature oocytes removal cumulus oocyte complexes（COCs）. In vitro maturation experimented with modified TaM medium or modified TaM medium supplement with L-Carnitine 1mM, 2mM, 5mM, and 10mM. In vitro maturation oocytes fertilized sperm of ICR. In vitro maturation medium supplement with L-Carnitine of maturation rate was 82 to 90％. There was not significant differential L-Carnitine non supplement. In vitro fertilization rate of L-Carnitine supplement was 67 to 80％.There was not significant differential L-Carnitine non supplement. In vitro early embryos development resulted 26％（129/499）, 27％（86/323）, 46％（136/293）, 37％（101/272） and 24％（71/302） respective. Moreover, Supplement withL-Carnitine 2mM and 5mM of blastcyst rate was significant higher than L-Carnitine 0mM of blastcyst rate （p＜0.05）. In conclusion, in vitro maturation medium supplement with appropriate L-Carnitine suggested that blastcyst rate was improvement.

[要約] 本実験では、麻酔下による外科的処置により卵巣摘出を施すことで、動物個体を処分することなく一部の卵巣組織のみを低侵襲的に回収し、そこから得られる未成熟卵子の発生能を検討した。B6D2F1（BDF1）系およびICR系マウスへPMSGを7.5単位投与し48時間後に麻酔処置をおこなった後、供試雌の卵巣を一部切除した（約2mm角）。回収した卵巣の胞状卵胞より、GV 期の未成熟卵子を回収しmTaM培地を用いて体外成熟および体外受精をおこなったところ、いずれの系統においても成熟培養後M II期卵子への発生を認め、体外受精成績はいずれも65％以上であった。またいずれの系統においても胚盤胞期胚までの発生を認めた。さらに、卵巣切除を施した各系統マウスにおいて、卵巣切片による卵胞観察像により卵子が確認され、自然交配により産子作出が可能であった。以上の結果より、外科的処置による卵巣の低侵襲的回収技術により得られた未成熟卵子由来の体外受精は可能であり、今後、低繁殖能を呈する系統や遺伝子改変マウスあるいは野生小型げっ歯類などの希少な動物個体を処分することなく、遺伝資源の効率的な保存・増殖方法の提示が期待されると示唆された。 [Abstract] This study, the developmental potential of immature oocytes collected low invasive by the surgical manipulation under the anesthesia was examined. Mice were used B6D2F1（BDF1）strain and ICR strain of matured mice. Mice are injected PMSG. After 48 hours, a part of ovaries（about 2mm）were extirpated under the anesthesia. Oocytes were collected from a part of ovary collected under the anesthesia Oocytes are transferred maturation medium（mTaM with 5％FBS）and matured in 5％ CO_2 incubator for 16 hours. In vitro matured oocytes were drilled by laser optimal system（XYClone； Nikko-Hansen Co., Ltd.）. In vitro matured oocytes drilled zona pellucida was performed in vitro fertilization and confirmed embryonic development. In vitro fertilization rate that used in vitro matured oocytes was over 65％ in each mice strain. Embryo development was developed to the blastocyst stage in each mice strain. In addition, oocytes were confirmed in the follicle images of the ovary slice after it had passed between convalescences in each mice strain removed the ovary.

[要約] 本研究では、マンモス骨髄組織からの効率的な細胞核の回収方法を開発するために、組織の状態が類似したウシ凍結骨髄組織からの効率的な細胞核の回収方法の開発を試みた。さらに、回収方法の有用性を検討するために、回収したウシ凍結骨髄組織由来細胞核をマウス除核未受精卵子に注入し、回収された核の生物学的特性の有無を検討した。 初めに、凍結保存されたウシ骨髄組織からの効率的な細胞核の回収方法の開発を試みた。ウシ骨髄組織から細胞核を回収する際に、Collagenase 処理を行う事によって、細胞核の回収量が有意に向上した。次に、回収したウシ凍結骨髄組織由来ドナー細胞核をマウス除核未受精卵子へ注入し、再構築卵子の早期染色体凝集および前核様構造物の確認を行った。その結果、早期染色体凝集ならびに前核様構造物の形成は観察されなかったものの、細胞核の注入直後に観察されたPropidium iodide による強い赤色蛍光が、細胞核注入後7 時間ではほぼ消失していた事から、生物学的特性は保持されていると考えられた。以上の結果より、生物学的特性を保持した状態の細胞核を大量に回収できる新しい細胞核の回収方法を開発する事に成功した。 [Abstract] We tried to develop the effective nuclei recovery method from cryopreserved cattle bone marrow tissues for the development of effective nuclei recovery method from mammoth bone marrow tissues. In addition, we studied whether its cell nuclei still kept their biological characteristics by injecting recovered nuclei into mouse enucleated matured oocytes. First, we tried to develop the effective nuclei recovery method from cryopreserved cattle bone marrow tissues. The amount of recovered cell nuclei was significantly increased by using collagenase treatment. Subsequently, we tried to inject recovered cell nuclei into mouse enucleated matured oocytes and checked for premature chromatin condensation and pronuclear-like structure. As a result, there was no oocyte with premature chromatin condensation and pronuclear-like structure, but because the highly-red fluorescence which was observed shortly after injection nearly disappeared after 7 hours, their cell nuclei were shown to keep biological characteristics. Based on these results, our method was shown to be effective in mass recovery of cell nuclei that kept their biological characteristics.

Gene therapy for dominantly inherited diseases with small interfering RNA (siRNA) requires mutant allele-specific suppression when genes in which mutation causes disease normally have an important role. We previously proposed a strategy for selective suppression of mutant alleles; both mutant and wild-type alleles are inhibited by most effective siRNA, and wild-type protein is restored using mRNA mutated to be resistant to the siRNA. Here, to prove the principle of this strategy in vivo, we applied it to our previously reported anti-copper/zinc superoxide dismutase (SOD1) short hairpin RNA (shRNA) transgenic (Tg) mice, in which the expression of the endogenous wild-type SOD1 gene was inhibited by more than 80%. These shRNA Tg mice showed hepatic lipid accumulation with mild liver dysfunction due to downregulation of endogenous wild-type SOD1. To rescue this side effect, we generated siRNA-resistant SOD1 Tg mice and crossed them with anti-SOD1 shRNA Tg mice, resulting in the disappearance of lipid accumulation in the liver. Furthermore, we also succeeded in mutant SOD1-specific gene suppression in the liver of SOD1(G93A) Tg mice, a model for amyotrophic lateral sclerosis, using intravenously administered viral vectors. Our method may prove useful for siRNA-based gene therapy for dominantly inherited diseases.

Curved DNA structures with a left-handed superhelical conformation can activate eukaryotic transcription. However, their potency in transgene activation in embryonic stem (ES) cells has not been examined. T20 is an artificial curved DNA of 180 bp that serves as a transcriptional activator. We investigated the effect of T20 on transcription in mouse ES cell lines or hepatocytes differentiated from them. We established 10 sets of cell lines each harboring a single copy of the reporter construct. Each set comprised a T20-harboring cell line and a T20-less control cell line. Analyses showed that in ES cells and in hepatocytes originating from these cells, T20 both activated and repressed transcription in a manner that was dependent on the locus of reporter. The present and previous studies strongly suggest that in cells that have a strict gene regulation system, transcriptional activation by T20 occurs only in a transcriptionally active locus in the genome.

We investigated the contribution of phosphorylated RNA polymerase II (RNAP II) and dynamic epigenetic changes to the onset of minor zygotic gene activation (ZGA) Using immunofluorescence staining, we observed that the nuclear Localization of RNAP IT was initiated by 6 hours post insemination (hpi), whereas RNAP II phosphorylated at serine residue 5 of the carboxyl-terminal domain (CTD) was localized by 9 hpi, and then RNAP II phosphorylated at serine residue 2 of the CTD was localized in the nucleus of embryos by 12 hpi In a transient gene expression assay using a plasmid reporter gene (p beta-actin/luciferase+/SV40) injected during 6-9 hpi into the male pronucleus, the luciferase+gene was actively transcribed and translated by 13 and 15 hpi, respectively, indicating that a transcriptionally silent state persisted for it least 4 hours after injection We found that the methylation status in the chicken beta-actin promoter region of the plasmid reporter gene may not be associated with the transcriptionally silent state before minor ZGA Exposure to trichostatin A did not induce premature expression of the silent reporter gene injected into 1-cell embryos containing histone deacetylase activity and did not affect the amount of luciferase produced per embryo Acetylated histone H3 lysine 9/14 and acetylated histone H4 lysine 12 and 16 were enriched preferentially in the injected reporter gene at least until 13 hpi, which coincided with the transcriptionally active state Taken together, these results suggest that deposition 01 selectively acetylated histones onto the chromatin of 1-cell embryos functions together with transcriptional elongation by RNAP II and that this sequential chromatin remodeling is involved in the molecular mechanism associated with the onset of minor ZGA in the preimplantation mouse embryo

In mammalian oocytes, the ubiquitin-proteasome system (UPS) is suggested to play important roles in oocyte meiosis resumption, spindle assembly, polar body emission and pronuclear formation by regulating cyclin B1 degradation Ho Never, little is known about the direct relationship between zygotic gene activation (ZGA) and degradation of maternal proteins Here, we investigated the role of the UPS in the onset of ZGA in early mouse embryo First, we found degradation of cyclin B1 protein in fertilized oocytes at 1 hpi by western blot analysis and used the se oocytes throughout this study Subsequently, we determined optimal experimental conditions for transient inhibition of proteasomal activity by specific and reversible proteasomal inhibitor MG132 in the Cl phase of the first cell cycle Under the selected optimal conditions, we subjected transient MG132-treated embryos to reverse transcription (RT)-PCR analysis of expression of four ZGA genes, i e, the hsp70 1, MuERV-L, eif-1a and zscan4d genes As a result, we found that onset of expression of the four examined ZGA genes was delayed m both normally developed 2-cell embryos and arrested 1-cell embryos Our results indicate that proteasomal degradation of proteins by the UPS plays a pivotal role in the molecular mechanisms of ZGA in early mouse embryos

We previously showed that circadian genes clock, bmal1, cry1, cry2, per1, and per2 are expressed and function as maternal mRNA regulating events in the oocytes and preimplantation embryos of mice. Recent evidence indicates however that either or both expression profiles of circadian genes in some tissues, and transcript sequences of circadian genes, differ to generate the physiological differences between diurnal and nocturnal species. We therefore investigated the expression profiles of circadian genes in oocytes and preimplantation embryos of species other than mice, namely cattle and rabbits, representing diurnal and nocturnal species, respectively, and determined the protein sequences of circadian genes in these species. Quantitative real-time PCR revealed that all circadian genes considered in this study were present in the oocytes and preimplantation embryos of both species, and the transcript amounts of clock, cry1 and per1 contained in oocytes were significantly higher than in preimplantation embryos of both species. The transcripts of clock, cry1, and per1 of cattle and rabbits were determined by primer walking, and functional domains in the estimated amino acid sequences were compared between cattle and rabbits and with those of humans and mice. The sequences of clock, cry1, and per1 in cattle and rabbits closely resembled those in mice (85-100% homologies), and no difference based on diurnality or nocturnality was observed. These findings suggest that circadian genes in the oocytes and preimplantation embryos of mammals fulfill the same functions across species as maternal mRNA. (C) 2010 Elsevier B.V. All rights reserved.

Recent studies have illustrated multiple differentiation potentials of embryonic stem cells (ESCs), derived from parthenogenetic embryos, to various kinds of cells (all three embryonic germ layers). However, differentiation diversity of the parthenogenetic ESCs (PgESCs) in vivo remains to be elucidated. In the present study, we established mouse PgESC-lines and observed their contribution diversity in vivo by producing chimeric mice using embryos possessing single nucleotide polymorphisms of mitochondrial DNA (mtDNA) as hosts. Based on southern blot analysis using specific probes to detect the SNPs on mtDNA, PgESC-derived mtDNA were contained in many organs such as brain, lung, and heart of the chimeric mouse. We concluded that PgESCs contributed to various internal organs in vivo, and that they were also stably maintained in adult animals. (C) 2010 Elsevier Inc. All rights reserved.

本実験では成熟齢C57BL/6J マウスから得られた体外成熟卵子を作出し、レーザー穿孔処理法を用いて各出力条件下（200, 150, 120μsec.）で透明帯穿孔処理を行ない、その後の体外受精および発生能を検討した。C57BL/6J 卵巣より回収した未成熟卵子の体外成熟成績は、91％（1, 517/1, 674）であった。レーザー穿孔処理時間による受精成績は、それぞれ、60％（191/316）, 54％（103/192）, 45％（196/439）であり、対照区（28％：129/463）と比較し有意な差が認められた（P<0.05）。また一部を培養した結果、胚盤胞期への発生率は31％（32/102）, 51％（74/144）, 53％（40/75）であった。２細胞期胚を移植した結果、レーザー出力を低出力にした場合、産子の発生向上が確認された〔6％（5/81）, 13％（10/83）, 21％（12/56）〕。さらにレーザー照射による熱変性を避けるため、卵細胞質を収縮させた透明帯穿孔卵子における受精成績も同様に対照区と比較し有意に向上した（p<0.05）。以上の結果より、C57BL/6J 未成熟卵子の体外成熟およびレーザー穿孔処理の条件を調整することにより、産子への発生を改善することが示唆された。

本研究では、マウスの耐凍剤を用いずに凍結された骨髄組織から回収された細胞を用いて体細胞核移植（somatic cell nuclear transfer; SCNT）を行い、永久凍土中から発見される動物遺物のような、耐凍剤を用いずに非常に長期間、不安定な環境下で凍結保存された生物の細胞をSCNT に用いることが可能か否か検討した。まず、耐凍剤を用いずに凍結保存されたマウスの骨髄細胞の回収と、DNA の断片化状態を確認した。回収された骨髄細胞をHoechst 33342 で染色し観察したところ、核の存在が確認された。コメットアッセイ及びフローサイトメトリーの結果から、耐凍剤を用いずに凍結保存された骨髄細胞では、多くの細胞のDNA が断片化していることが確認された。次に、回収した骨髄細胞を、核ドナー細胞としてSCNT に用いた。その結果、マウス新鮮骨髄細胞および−80℃凍結骨髄細胞を用いた再構築卵子が、胚盤胞期にまで発生した（16.9％、及び1.9％）。以上の結果より、耐凍剤を用いずに凍結保存された骨髄細胞は、凍結保存されることにより、DNA に大きな障害を受けていることが確認されたが、SCNT の核ドナー細胞として利用できる可能性も示された。

Here, we report the recovery of cell nuclei front 14,000-15,000 years old mammoth tissues and the injection of those nuclei into mouse enucleated matured oocytes by somatic cell nuclear transfer (SCNT). From both skin and muscle tissues, cell nucleus-like structures were successfully recovered. Those nuclei were then injected into enucleated oocytes and more than half of the oocytes were able to survive. Injected nuclei were not taken apart and remained its nuclear structure. Those oocytes did not show disappearance of nuclear membrane or premature chromosome condensation (PCC) at 1 hour after injection and did not form pronuclear-like structures at 7 hours after injection. As half of the oocytes injected with nuclei derived from frozen-thawed mouse bone marrow cells were able to form pronuclear-like structures, it might be possible to promote the cell cycle of nuclei from ancient animal tissues by suitable pre-treatment in SCNT. This is the first report of SCNT with nuclei derived from mammoth tissues.

The embryos of some rodents and primates can precede early development without the process of fertilization; however, they cease to develop after implantation because of restricted expressions of imprinting genes. Asexually developed embryos are classified into parthenote/gynogenote and androgenote by their genomic origins. Embryonic stem cells (ESCs) derived from asexual origins have also been reported. To date, ESCs derived from parthenogenetic embryos (PgESCs) have been established in some species, including humans, and the possibility to be alternative sources for autologous cell transplantation in regenerative medicine has been proposed. However, some developmental characteristics, which might be important for therapeutic applications, such as multiple differentiation capacity and transplantability of the ESCs of androgenetic origin (AgESCs) are uncertain. Here, we induced differentiation of mouse AgESCs and observed derivation of neural cells, cardiomyocytes and hepatocytes in vitro. Following differentiated embryoid body (EB) transplantation in various mouse strains including the strain of origin, we found that the EBs Could engraft in theoretically MHC-matched strains. Our results indicate that AgESCs possess at least two important characteristics, multiple differentiation properties in vitro and transplantability after differentiation, and suggest that they can also serve as a source of histocompatible, tissues for transplantation.

Oct-4 is essential for normal embryonic development, and abnormal Oct-4 expression in cloned embryos contributes to cloning inefficiency. However, the causes of abnormal Oct-4 expression in cloned embryos are not well understood. As DNA methylation in regulatory regions is known to control transcriptional activity, we investigated the methylation status of three transcriptional regulatory regions of the Oct-4 gene in cloned mouse embryos-the distal enhancer (DE), the proximal enhancer (PE), and the promoter regions. We also investigated the level of Oct-4 gene expression in cloned embryos. Immunochemistry revealed that 85% of cloned blastocysts expressed Oct-4 in both trophectoderm and inner cell mass cells. DNA methylation analysis revealed that the PE region methylation was greater in cloned morulae than in normal morulae. However, the same region was less methylated in cloned blastocysts than in normal blastocysts. We found abnormal expression of de novo methyltransferase 3b in cloned blastocysts. These results indicate that cloned embryos have aberrant DNA methylation in the CpG sites of the PE region of Oct-4, and this may contribute directly to abnormal expression of this gene in cloned embryos.

In mammals, circadian genes, Clock, Arntl (also known as Bmal1), Cry1, Cry2, Per1, Per2, and Per3, are rhythmically transcribed every 24 h in almost all organs and tissues to tick the circadian clock. However, their expression and function in oocytes and preimplantation embryos have not been investigated. In this study we found that the circadian clock may stop in mouse oocytes and preimplantation embryos. Real-time PCR analysis revealed the presence of transcripts of these genes in both oocytes and preimplantation embryos; however, their amounts did not oscillate every 24 h in one- to four-cell and blastocyst-stage embryos. Moreover, immunofluorescence analyses revealed that CLOCK, ARNTL, and CRY1 were localized similarly in the nuclei of germinal vesicle (GV) oocytes and one-cell- to four-cell-stage embryos. Because CRY1 is known to interact with the CLOCK-ARNTL complex to suppress transcription-promoting activity of the complex for genes such as Wee1, Cry2, Per1, Per2, and Per3 in cells having the ticking circadian clock, we hypothesized that if the circadian clock functions in GV oocytes and one-cell- to four-cell-stage embryos, CLOCK, ARNTL, and CRY1 might suppress the transcription of these genes in GV oocytes and one-cell- to 4-cell-stage embryos as well. As a result, knockdown of CRY1 in GV oocytes by RNA interference did not affect the transcription levels of Wee1, Cry2, Per1, Per2, and Per3, but it reduced maturation ability. Thus, it seems that circadian genes are not involved in circadian clock regulation in mouse oocytes and preimplantation embryos but are involved in physiologies, such as meiosis.

体細胞核移植 (Somatic cell nuclear transfer: SCNT)技術は、1997年のWilmutらによる報告後、様々な動物種においてクローン個体の作出を実現してきたが、10余年を経た現在においてもその作出効率は未だに低率である。その原因を解明し作出効率を上げるための研究は活発に行われているが、その根本的な原因は不明なままである。近年、生細胞における分子の動態や、クロマチンの配置などの解析から、核内構造構築が転写活性などの遺伝子発現制御の基盤となることがわかってきた。そして最近、核内のアクチン関連タンパク質 (Arp) がクロマチン構造をする複合体に広く含まれており、Arpファミリータンパク質が細胞核、クロマチンの機能構造やダイナミクスに関与していることが示唆されている。本研究では、受精卵および核移植由来卵子において、クロマチンリモデリング因子SWR1複合体の構成因子であるArp4、Arp6、SWR1の局在を免疫組織化学的染色によって解析した。その結果、受精卵および核移植由来卵で、クロマチン構造に違いが見られた領域でのArp4およびSWR1の発現パターンが異なっていた。このことから、クロマチン配置の相違、あるいは核内構造制御タンパク質の局在パターンが核移植由来卵子での核の不適切なリプログラミングを反映している可能性が示唆された。

In short hairpin RNA (shRNA) transgenic mice, the tissue difference in gene silencing efficiency and oversaturation of microRNA (miRNA) pathway have not been well assessed. We studied these problems in our previously-reported anti-copper/zinc superoxide dismutase (SOD1) shRNA transgenic mice. Although there was a tissue difference (liver and skeletal muscle, >95%; central nervous system and lung, similar to 80%), the target gene silencing was systemic and our anti-SOD1 shRNA transgenic mice recapitulated the SOD1-null mice. Neither endogenous miRNAs nor their target gene levels were altered, indicating the preservation of endogenous miRNA pathways. We think that the shRNA transgenic mice can be utilized for gene analysis. (C) 2008 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Primordial germ cells (PGCs) are embryonic precursors of the gametes of adult animals and are considered stem cells of the germline. Since their proliferation in vitro correlates well with the schedule of developmental changes in vivo, they might be interesting research tools for genomic imprinting, germ-cell tumors and fertility. Furthermore, once primordial germ cells are separated and placed on a feeder layer with cytokines, they become Cultured pluripotent cell lines called embryonic germ (EG) cells. EG cells share several important characteristics with embryonic stem (ES) cells as they call also contribute to the germ line of chimeras. To investigate the characteristics of PGCs and establish rabbit EG (rEG) cells, we cultured rabbit PGCs (rPGCs) in vitro with various combinations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and forskolin oil inactivated mouse embryonic fibroblast (MEF) feeder layers. The present study found PGC proliferation in early cultures and induction of rEG-like colonies. These cells expressed pluripotent markers, such as alkaline phosphatase activity, OCT-4, Sox-2 and SSEA-1, in the undifferentiated state; however, the cells did not develop into a teratoma when injected into the kidney capsules of SCID mice, although the restricted differentiation potentials to neural cells were determined via embryoid body formation. From these characteristics and further characterization of the germ stem cell markers Vasa, SCP-1 and SCP-3, we suggested that these were hybrid cells with characteristics somewhere between PGC and EG cells.

We examined the promoter activities of three mouse maternal genes (H1oo, Npm2, and Zar1) in oocytes and pre-implantation embryos, and examined the promoters for cis-acting elements of 5'-flanking region to obtain the best promoter for inducing oocyte-specific gene expression. For the assay, we injected firefly luciferase gene constructs under the control of the promoters into the oocytes and embryos. Each promoter region showed transcriptional activity in oocytes, but not in fertilized embryos. Deletion analysis showed that a putative E-box region at position -72 of the H1oo promoter and at the -180 of the Npm2 promoter were required for basal transcriptional activity in oocytes. Moreover, a putative NBE motif (NOBOX DNA binding elements) (-1796) was shown to enhance basal transcriptional activity of the Npm2 promoter. Thus, the E-box and/or NBE may be key regulatory regions for the expression of the examined maternal genes (H1oo and Npm2) in growing mouse oocytes.

We isolated a mouse cDNA, zag1 (zygotic gene activation-associated gene 1), that has an open reading frame of 1,728-bp encoding a protein of 66.2 kDa including both a bipartite nuclear targeting sequence and a P-loop motif containing nucleoside triphosphate hydrolase motifs. Northern blot analysis of mouse tissues showed that zag1 was widely expressed but was especially prominent in the ovary and testis. RT-PCR analysis of in vitro fertilized embryos showed that the abundance of zag1 transcripts in oocytes decreased after fertilization, and zag1 mRNA was detected at 15 h post insemination (hpi) in fertilized embryos indicating that the gene was expressed at the start of zygotic gene activation at the mouse 1-cell stage. The nuclear-localization of ZAG1 protein in mouse preimplantation embryos at 15 hpi was confirmed by both subcellular analysis of enhanced green fluorescent protein (EGFP)-tagged ZAG1 and immunocytochemical analysis with anti-ZAG1 antibody. Subsequently, using yeast two-hybrid screening, we identified U2 small nuclear ribonucleoprotein B (U2B"), which is associated with pre-mRNA splicing, as a putative interacting partner of ZAG1 protein. Furthermore, knockdown of zag1 expression by an antisense DNA plasmid induced arrest and/or delay of embryonic development in injected 1-cell embryos. These results suggest that ZAG1 may be closely associated with zygotic gene expression in mouse preimplantation embryos.

げっ歯類のゲノム中には、Intracisternal A Particle（IAP）と呼ばれるレトロトランスポゾンが存在する。IAP は、その両端にLong Terminal Repeat（LTR）と呼ばれる、強力な転写プロモーター活性やエンハンサー活性を持つ、長い末端反復配列を持つ（Dewannieux et al., 2004）。このLTR 配列の転写プロモーター活性やエンハンサー活性は、ゲノム中のIAP 挿入部位の前後に存在する遺伝子の発現に影響を及ぼすことが報告されている（Barbot et al., 2002, Jaenisch et al.,2003, Morgan et al., 1999）。実験動物であるマウスにおいては、IAP を含むレトロトランスポゾンについて多くの研究が報告されているが、家畜であるウシにおいては、レトロトランスポゾンに関する研究はほとんどなされていない。ウシゲノムプロジェクトが進行中である現在、ウシゲノムにおけるトランスポゾンなどの機能未知であるnon-coding 領域の研究を行うことは、ウシゲノムの機能解析においても有効であると考えられる。そこで本研究では、当研究室において得られたマウスIAP 様ウシレトロトランスポゾンのLTR 配列を転写プロモーターとして持つ、Enhanced Green Fluorescent Protein（EGFP）発現ベクターを構築し、マウス線維芽細胞、ウシ線維芽細胞およびマウスES 細胞へ導入し、ウシゲノム中に存在するマウスIAP 様ウシレトロトランスポゾンのLTR 配列の転写プロモーター活性について検討した。

本研究では、マウス体細胞核移植（Somatic Cell Nuclear Transfer : SCNT）胚の産子への発生能の検討と作出されたクローンマウスの生育ならびに繁殖能力について検討した。ドナー細胞として卵丘細胞を用いて核移植を行い、得られた2 細胞期の再構築胚を卵管へ移植した。その結果、1 匹のクローン産子を作出することに成功した（生存産子1 匹/ 移植胚数24 個：4.2％）。クローンマウスの生殖能力について検討した結果、2 度の自然分娩で計20 匹の産子を獲得し、正常に子孫を得ることが確認された。また、クローンマウスとその産子それぞれについて経時的に体重測定を行った結果、クローンマウスで従来報告されているような極度の肥満はみられず、またその産子では肥満はみられなかった。さらに、各近交系間のサテライトマーカーを用いた遺伝的背景の解析を行った結果、クローンマウスの遺伝的背景はドナー細胞のB6D2F1 と一致した。これらの結果から、B6D2F1 卵丘細胞由来の体細胞クローンマウスでは、正常な繁殖能力を有し肥満もみられないこと、マイクロサテライトマーカーでの解析においては、遺伝的背景はドナー細胞により決定されることが示された。

近年、マウスやラットにおいて精子形成を通して次世代の個体構築を担う唯一の幹細胞である精原幹細胞を体外で培養し、株化した生殖幹細胞（Germline Stem Cell；GS 細胞）が樹立された。しかし、ウシなどの食資源動物ではES 細胞やEG 細胞はいまだ樹立されておらず、高度な遺伝子改変を行う上で、代替えとなる幹細胞の樹立が望まれる。ウシ雄性生殖細胞の同定には、ヘマトキシリン・エオシン染色やレクチンの一種であるDBA やc-kit を用いた組織学的解析やフローサイトメトリー（FCM）による解析が主に行われてきた。これにより、ウシ精巣内のゴノサイトや精原幹細胞やA 型精原細胞が、同定されてきた。そこで本研究では、ウシにおけるゴノサイトおよび精原幹細胞の同定を目的として、RT-PCR ならびにIn situ hybridization 法により、未分化マーカーとされているOct3/4 のmRNA の検出を試みた。さらに、ゴノサイトで発現しているVASA タンパク質に注目し、月齢の異なるウシ精巣内、ならびに初代培養したin vitro でのゴノサイトの局在を免疫組織化学染色により同定した。その結果、ウシOct3/4 は、RT-PCR において分化細胞からも検出され、さらにIn situ hybridization においても、精細管内で特異的な発現は認められなかった。一方、VASA タンパク質は若齢ウシ精巣においてマウスと同様の発現パターンを示し、またウシ精巣細胞の初代培養においてもVASA 陽性細胞の増殖が認められた。これらの結果から、ウシ若齢精巣において、VASA はゴノサイトや精原幹細胞で発現し、体外培養法の開発にも有効であることが示された。

近年、幹細胞が蛍光色素であるHoechst33342 を強力に排出する性質を示すということを利用した、新たな幹細胞の識別方法が報告されている。この方法により分離される幹細胞は、フローサイトメトリーにおいて SP（ Side-Population）分画として検出される。このような、幹細胞のHoechst33342 排出能力に関与する遺伝子として、ABC（ATP-binding cassette）トランスポーターファミリーのひとつであるBcrp1（Breast cancer resistance protein 1）が報告された。さらに近年、Bcrp1 Exon1 には、3 つのアイソフォーム（A、B およびC）が存在し、造血幹細胞においては分化段階に応じてBcrp1 mRNA アイソフォームの転写に選択性があることが示された。そこで本研究では、まずマウス組織ならびに培養細胞におけるBcrp1mRNA アイソフォームの選択性について検討するとともに、未分化マウスES 細胞におけるBcrp1 mRNAアイソフォームの発現量をReal-time PCR により定量的に解析した。その結果、アイソフォームA が最も高く発現し、アイソフォームC の発現はきわめて低いことが示された。さらに、分化誘導過程におけるBcrp1 mRNA アイソフォームの発現について検討した結果、未分化ES 細胞で高発現しているアイソフォームA およびB は、Oct3/4 やNanog の発現低下に先立ち、分化誘導初期に一度急激に減衰し、その後再び発現が回復することが示された。以上の結果より、Bcrp1 mRNA アイソフォームの発現と選択性はES 細胞の分化状態に何らかの影響をもたらすことが示唆された。

胚性幹細胞（Embryonic Stem Cell ; ES 細胞）は自己複製能と様々な細胞へと分化することの出来る分化多能性を有している細胞である。近年、このES 細胞を用いた再生修復医療の可能性が注目されている。しかし、受精卵からES 細胞を作出するため、移植後に免疫拒絶反応を示すことが示唆されており、ES 細胞を用いた再生修復医療の1 つの課題となっている。そこで、片親性の雌性単為発生胚と雄性発生胚からES 細胞を樹立し、片親性ES 細胞の分化多能性を観察した。

The majority of somatic cell nuclear transferred (SCNT) embryos die before or after implantation. Many studies have focused on morphological remodeling of the donor nucleus and its associated cytoskeletal structures in the early events of nuclear transfer. However, little is known about the 2-cell stage of SCNT embryos after the first division. In this study, we compared the morphological status of chromosomal division during the 1-cell stage to the 2-cell stage in SCNT embryos with that in intracytoplasmic sperm injection (ICSI) embryos. The microtubules and cytoplasmic asters, which are related to chromatin segregation, disappeared at the pronuclear stage, although formation of the first mitotic spindle was normal in both the SCNT and ICSI embryos. However, nuclear fragmentation was observed in 30% of the 2-cell SCNT embryos and 12% of the 2-cell ICSI embryos. Nuclear fragmentation was present in both blastomeres of these embryos. No apoptotic DNA fragmentation was observed in TdT-mediated dUTP-biotin Nick End Labeling (TUNEL) assays for either the SCNT or ICSI embryos. In both the SCNT and ICSI embryos, the distribution of chromosomes in the first mitotic spindle was disturbed during the process of division from the 1-cell stage to the 2-cell stage. These results suggest that loss of SCNT embryos just before or after implantation may be due to an abnormal chromosome distribution at the 2-cell stage.

CpG 配列におけるシトシン残基のメチル化はゲノムにおける主要なエピジェネティックな修飾であり、遺伝子発現の制御に重要な役割を演じている。 近年、ゲノム中の全体的なDNA のメチル化状態は、薄層クロマトグラフィー、Restriction Landmark Genomic Scanning（RLGS）法やRepresentational Difference Analysis（RDA）法によって研究されている。 しかしながら、これらの方法は複雑であり、制限酵素によるゲノムの処理を必要とする。それゆえに、これらの方法から得られる情報は、使用した制限酵素によって切断できるDNA 塩基配列に限られている。 本研究では、ウシ精子における全体的なDNA メチル化の状態を解析する簡便な方法を確立するために、メチル化シトシンを免疫蛍光抗体染色する方法をウシ精子DNA に用いる方法の開発を試みた。 本研究中のメチル化シトシンの免疫蛍光抗体染色法は、Benchaib ら（2003）がヒト精子を用いて開発した方法を基にした。種間差により、Benchaib らによって開発された方法をウシ精子に用いてもメチル化シトシンの検出ができなかったため、方法中の手技を幾つか変更した。ウシ凍結融解精子はパーコール洗浄によって卵ク液および混入体細胞を分離した。洗浄後、精子を0.25M DTT と1％ SDS を用いて 室温にて処理した。処理精子をサイトスピン4 を用いてスライドガラス上へ展開し（30 × g, 5 × 10^4 cells/ml） 室温にて風乾した。風乾した精子標本を室温にてメタノール：氷酢酸（3 : 1）の固定液へ浸漬して固定し、その後、室温にて1％ Triton X と1％ SDS を用いて処理した。処理後、DNA を6N HCl を用いて変性し、精子DNA 中のメチル化シトシンを免疫蛍光抗体染色法によって染色し、共焦点レーザー顕微鏡を用いて画像を撮影した。撮影された画像を画像解析ソフトウェアによって解析した。 精子頭部中、ヨウ化プロピジウムによって染色された領域を頭部DNA の領域とし、その領域中に存在するFITC の蛍光を示す領域をメチル化シトシンの領域とし、それぞれの領域の面積を画像解析ソフトウェアによって計測し、その面積比を算出した。面積の計測は一定の紫外線強度の下で行われた。精子頭部中の全体のDNA 面積に対するメチル化シトシンの面積比は、種雄牛A においては34.6％（9.98 ± 5.39μm^2, 28.25 ± 4.98μm^2, n=248）であり、種雄牛B においては39.3％（12.29 ± 5.39μm^2, 31.74 ± 5.96μm^2, n=165）、また種雄牛C においては40.6％（13.67 ± 4.84μm^2, 33.70 ± 5.69μm^2, n=46）であった。 種雄牛A と、種雄牛B およびCの精子頭部のDNA メチル化状態には有意な差が存在した（P＜0.01）。この種雄牛間のシトシンのメチル化状態の差異が意味することについては、今後の詳細な研究が必要である。

体細胞核移植（somatic cell nuclear transfer : SCNT）技術は、1997 年のWakayama らによる報告以来、様々な動物種においてクローン個体の作出を実現してきたが、その作出効率は未だに低率である。マウスにおいては、核移植胚のおよそ半分は着床するが、出生まで至るものはきわめて少ない。着床の成立と維持における胚側の要素として、栄養外胚葉系譜への分化は必須の過程であるが、最近、マウス栄養芽細胞で転写因子、Caudal-related homeobox 2 （Cdx2）が発現し、栄養外胚葉系譜の誘導で重要な役割を果たしていることが報告された。さらに、Cdx2 は、初期胚発生過程でOct3/4 と互いに制御関係にあることが示された。そこで本研究では、着床、胎盤形成の基点となる栄養外胚葉で発現するCdx2 に着目し、Cdx2 とその制御関係にあるOct3/4 について、マウスSCNT胚と顕微授精（ICSI）胚での発現解析を行った。その結果、SCNT胚において、Cdx2 は発現しているものの、ICSI 胚に比べ転写量は約半分と低かった。このことから、Cdx2 の発現の低下が、その後の栄養外胚葉系譜での遺伝子発現に影響し、着床の成立と維持に障害をもたらしている可能性が示唆された。

Embryonic stem cells (ESCs) of nonhuman primates are important for research into human gametogenesis because of similarities between the embryos and fetuses of nonhuman primates and those of humans. Recently, the formation of germ cells from mouse ESCs in vitro has been reported. In this study, we established cynomolgus monkey ES cell lines (cyESCs) and attempted to induce their differentiation into germ cells to obtain further information on the development of primate germ cells by observing the markers specific to germ cells. Three cyESCs were newly established and confirmed to be pluripotent. When the cells are induced to differentiate, the transcripts of Vasa and some meiotic markers were expressed. VASA protein accumulated in differentiated cell clumps and VASA-positive cells gathered in clumps as the number of differentiation days increased. In the later stages, VASA-positive clumps coexpressed OCT-4, suggesting that these cells might correspond to early gonocytes at the postmigration stage. Furthermore, meiosis-specific gene expression was also observed. These results demonstrate that cyESCs can differentiate to developing germ cells such as primordial germ cells (PGCs) or more developed gonocytes in our differentiation systems, and may be a suitable model for studying the mechanisms of primate germ cell development. © Mary Ann Liebert, Inc.

Development of assisted reproductive technologies is necessary to obtain fertilized oocytes in a subfertile transgenic mouse strain. Here, we showed the application of laser-assisted drilling of the zona pellucida to in vitro fertilization of cryopreserved mouse oocytes with sperm from subfertile transgenic mice (C57BL/6N-Tg(UCP/FAD2)U8 strain). After cryopreservation by vitrification, the recovery and survival rates of the zona-drilled mouse oocytes were 97% (97/100) and 94% (91/97), respectively. In vitro fertilization of the cryopreserved zona-drilled mouse oocytes with sperm from the subfertile transgenic mice was greatly facilitated (60%, 55/91) compared to that of the cryopreserved zona-intact mouse oocytes (11%, 81/768). In vitro fertilized embryos that developed to the 2-cell stage were again cryopreserved by vitrification, and after warming they were transferred into recipient females. Subsequently, six viable offspring were delivered, and all were confirmed to be transgenic mice. These results indicate that laser-assisted zona drilling of oocytes combined with cryopreservation by vitrification may be a useful approach for large-scale production of in vitro fertilized embryos for managing transgenic mouse strains with reproductive disabilities such as subfertile sperm.

Morpholino oligonucleotides (MO) can induce gene silencing by binding to a target mRNA and inhibiting its translation, and this technique has been especially successful in studies of embryonic development in various vertebrates. But in mice MO-induced downregulation of target genes has not been widely reported. In this study, we examined whether MO delivery using ethoxylated polyethylenimine (EPEI) delivery reagent is useful for silencing gene expression in the mouse preimplantation embryo, by targeting endogenous gene Oct4. To optimize the conditions for MO delivery, we examined the MO concentration, the EPEI concentration, the treatment time, and the number of MO treatments. The MO treatment was performed at the 2-cell, the morula, the blastocyst, and the hatched blastocyst stage. We first determined the optimal conditions for MO delivery into the nucleus using fluorescein isothiocianate (FITC)-labeled MO, and demonstrated that treatment with a combination of 20 mu M MO and 0.56 mu M EPEI for 3 hrs produced effective MO delivery. MO-induced downregulation of Oct4 was then examined. Two-step MO treatment at the 2-cell and blastocyst stages successfully suppressed Oct4 expression. This MO treatment resulted in marked reduction of Oct4 protein at the blastocyst stage. After cultivation of blastocysts for further 4 days, derivatives of embryos either differentiated to trophoblastic cells or showed developmental arrest at the blastocyst. This phenocopy is similar to Oct4-deficient embryos. Overall, our results indicate that MO delivery with EPEI is an effective tool for analyzing gene function in mouse preimplantation embryos.

哺乳動物において、その発生の途上において重要ないくつかの遺伝子は、ゲノム刷り込みの影響を受ける。本研究では、父親由来のゲノム刷り込みの個体発生に対する影響を検討する第一段階として、マウス雌雄体細胞、精子、卵子、野生型および雄性発生胚由来ES 細胞におけるH19 遺伝子の5 '上流に存在するDMR におけるCpG のメチル化の状態を解析した。その結果、雄性発生胚由来ES 細胞におけるDMR の5 '上流部分のCpG は、精子と同様に高度にメチル化されていた。しかしながら、DMR の3 ' 下流部分のCpGは、雄性発生胚由来ES 細胞において、既にそのメチル化パターンは、雄性配偶子である精子とは異なることが観察された。

Recently, studies on cell surface markers of spermatogonia in combination with germ cell transplantation technique have made possible their functional analysis and the germline stem cells（GS cell）have established. GS cells are downstream of the stem cells such as ES cells and embryonic germ cells, namely EG cells, which are derived from PGCs. Therefore, GS cells are expected to be useful for the production of genetically-modified animals. In this study, we examined enrichment of GS cells expressing CD9 or α6-integrin from C57BL/6J cryptorchid adult and ICR pup（6-8 dpp）testes by magnetic cell sorting（MACS）and cultivation of those cells in vitro. Flow cytometric analyses demonstrated that MACS effectively enriched CD9-positive（CD9^+）and α6-integrin-positive （α6-integrin^+）cells most effectively from pup testis. Therefore, CD9^+ and α6-integrin^+ cells from pup testes were used for the following cultivation. Cells proliferated in the first few days in suspension, subsequently attached to the culture plates and formed colonies after 3-5 days of culture. Those GS-like cells were passaged on the feeder layers and showed continuous proliferation for more than 2 months. Immunocytochemical and FCM analyses demonstrated that those cells maintained the expression of CD9, α6-integrin and Oct4. These findings indicate that the CD9^+ and α6-integrin^+ cells collected from mouse pup testes have GS cell properties. Now, transplantation of GS-like cells into testis of recipient W mice which lack spermatogenesis is under way to evaluate their ability of spermatogenesis.

Many autosomal dominant diseases such as familial amyotrophic lateral sclerosis (ALS) with copper/zinc superoxide dismutase (SOD1) mutation may be induced by missense point mutations that result in the production of proteins with toxic properties. Reduction in the encoding of proteins from such mutated genes can therefore be expected to improve the disease phenotype. The duplex of 21-nucleotide RNA, known as small interfering RNA (siRNA), has recently emerged as a powerful gene silencing tool. We made transgenic (Tg) mice with modified siRNA, which had multiple mismatch alternations within the sense strand, to prevent the "shutdown phenomenon" of transgenic siRNA. Consequently, the in vivo knockdown effect of siRNA on SOD1 expression did not diminish over four generations. When we crossed these anti-SOD1 siRNA Tg mice with SOD1(G93A) Tg mice, a model for ALS, siRNA prevented the development of disease by inhibiting mutant G93A SOD1 production in the central nervous system. Our findings clearly proved the principle that siRNA-mediated gene silencing can stop the development of familial ALS with SOD1 mutation.

ウシの体細胞による再構築胚の体外培養による発生率は未だ低い。本研究では、血清飢餓培養したウシ耳介由来繊維芽細胞を除核したウシ未受精卵に融合して作製した再構築胚を用いて融合後168 時間体外で培養し、効果的な培養方法を確立するために行った。実験I では、修正合成卵管液（mSOF）、アミノ酸を添加したCharles Rosenkrans 液（CR1aa）および5％ 新生子ウシ血清を添加したCR1aa （CR1aa + NBCS）で再構築胚を培養した。無血清培養液であるCR1aa 液では、胚盤胞期胚は得られなかったが、血清を添加したCR1aa+NBCS では、培養した再構築胚の13％ が胚盤胞期胚に発生した。一方、mSOF 液では、無血清培養液であるにもかかわらず17％ の胚盤胞への発生率が得られ、CR1aa+NBCS と同様だった（p ＜ 0.05）。実験II では、体細胞再構築胚を融合後120 時間までmSOF あるいはCR1aa+NBCS で培養し、その後細胞培養液である5％ NBCS を添加したダルベッコイーグル培養液（DMEM - NBCS）で168 時間まで培養した。その結果、2 区の培養方法でともに21- 25％ の再構築胚が胚盤胞期へ発生し、ウシ再構築胚を培養後半期にDMEM - NBCS で培養すると発生率が向上することが示された。

本研究では、核移植によるクローンマウスの作出を目的として卵丘細胞をドナー細胞に用いた核移植を行い、核移植胚の体外培養における発生能および胚移植後の発生能の検討を行った。胚移植の結果、着床痕が認められ、肥大した胎盤の形成を確認することができた。さらに、マウスのクローン産仔を得ることができた。

卵丘細胞を注入したマウス再構築胚発生において、再構築卵子活性化処理培地中へのジメチルスルフォキシド（DMSO : dimethyl sulfoxide）の添加が、その後の発生率に及ぼす影響について検討を行った。注入後の卵子は塩化ストロンチウム、サイトカラシンB 及び各濃度のDMSO（0.05,0.1,0.5,1,2,4％）含有のCa2^+ フリーmCZB 培地+0.3％ BSA で活性化処理を行った。活性化処理培地中へDMSO を添加した群において、未添加群と比較したところ、2 細胞期への発生率が改善されている傾向が認められた（2％：57％ ,未添加：36％）。また、DMSO の濃度は2％添加にて、桑実期及び胚盤胞期への発生率が改善される傾向が認められた。DMSO 未添加群と、2％添加群について、活性化処理後24 時間での再構築胚の断片化について検討したところ、2％添加群において、断片化の改善が認められた。以上の結果より、卵丘細胞を用いたマウス再構築胚の発生率及び断片化抑制について、DMSO は有効であることが示唆された。

Autoimmune regulator (AIRE) gene mutation is responsible for the development of organ-specific autoimmune disease with monogenic autosomal recessive inheritance. Although Aire has been considered to regulate the elimination of autoreactive T cells through transcriptional control of tissue-specific Ags in thymic epithelial cells, other mechanisms of AIRE-dependent tolerance remain to be investigated. We have established Aire-deficient mice and examined the mechanisms underlying the breakdown of self-tolerance. The production and/or function of immunoregulatory T cells were retained in the Aire-deficient mice. The mice developed Sjogren's syndrome-like pathologic changes in the exocrine organs, and this was associated with autoimmunity against a ubiquitous protein, a-fodrin. Remarkably, transcriptional expression of alpha-fodrin was retained in the Aire-deficient thymus. These results suggest that Aire regulates the survival of autoreactive T cells beyond transcriptional control of self-protein expression in the thymus, at least against this ubiquitous protein. Rather, Aire may regulate the processing and/or presentation of self-proteins so that the maturing T cells can recognize the self-Ags in a form capable of efficiently triggering autoreactive T cells. With the use of inbred Aire-deficient mouse strains, we also demonstrate the presence of some additional factor(s) that determine the target-organ specificity of the autoimmune disease caused by Aire deficiency.

Recently, with the acetylation of histone and the modification of chromatin structure, the methylation of cytosine residue within CpG dinucleotides in genomic DNA sequence attracts many researchers' attention as one of major epigenetic regulation systems of gene expression. There are several methods (immunofluorescence with 5-methylcytosine specific antibody and methylationsensitive restriction enzyme-PCR method, etc.) to analyze the methylation of cytosine residue. Bisulfitesequencing method, which is one of methods for analyzing the methylation of cytosine residue in genomic DNA sequence, has advantages of high sensitivity and analyzing the methylation of cytosine residue directly. In this note, the detailed procedure of bisulfite-sequencing method for mouse early Preimplantation embryos is described. © 2005, JAPANESE SOCIETY OF OVA RESEARCH. All rights reserved.

Nucling is a novel protein isolated from murine embryonal carcinoma cells with an up-regulated expression during cardiac muscle differentiation. We show here that Nucling was up-regulated by proapoptotic stimuli and important for the induction of apoptosis after cytotoxic stress. We further demonstrated that overexpressed Nucling was able to induce apoptosis. In Nucling-deficient cells, the expression levels of Apaf-1 and cytochrome c, which are the major components of an apoptosis-promoting complex named apoptosome, were both down-regulated under cellular stress. A deficiency of Nucling also conferred resistance to apoptotic stress on the cell. After UV irradiation, Nucling was shown to reside in an Apaf-1/pro-caspase-9 complex, suggesting that Nucling might be a key molecule for the formation and maintenance of this complex. Nucling induced translocation of Apaf-1 to the nucleus, thereby distributing the Nucling/Apaf-1/pro-caspase-9 complex to the nuclear fraction. These findings suggest that Nucling recruits and transports the apoptosome complex during stress-induced apoptosis.

Impaired insulin secretion and insulin resistance are thought to be two major causes of type 2 diabetes mellitus. There are two kinds of diabetic model mice: one is a K-ATP channel knockout (Kir6.2KO) mouse which is defective in glucose-induced insulin secretion, and the other is a transgenic mouse expressing the tyrosine kinase-deficient (dominant-negative form of) human insulin receptor (hIR(KM)TG), and which has insulin resistance in muscle and fat. However, all of these mice have no evidence of overt diabetes. To determine if the double mutant Kir6.2KO/hIR(KM)TG mice would have diabetes, we generated mutant mice by crossbreeding, which would show both impaired glucose-induced insulin secretion and insulin resistance in muscle and fat. We report here that: 1) blood glucose levels of randomly fed and 6 h fasted double mutant (Kir6.2KO/hIR(KM)TG) mice were comparable with those of wild type mice; 2) in intraperitoneal glucose tolerance test (ipGTT), Kir6.2KO/hIR(KM)TG mice had an impaired glucose tolerance; and 3) during ipGTT, insulin secretion was not induced in either Kir6.2KO/hIR(KM)TG or Kir6.2KO mice, while the hIR(KM)TG mice showed a more prolonged insulin secretion than did wild type mice; 4) hyperinsulinemic euglycemic clamp test revealed that Kir6.2KO, Kir6.2KO/hIR(KM)TG and hIR(KM)TG mice, showed decreased whole-body glucose disposal compared with wild type mice; 5) Kir6.2KO, but not Kir6.2KO/hIR(KM)TG mice had some obesity and hyperleptinemia compared with wild type mice. Thus, the defects in glucose-induced insulin secretion (Kir6.2KO) and an insulin resistance in muscle and fat (hIR(KM)TG) were not sufficient to lead to overt diabetes.

Physical contact between thymocytes and the thymic stroma is essential for T cell maturation and shapes the T cell repertoire in the periphery. Stromal elements that control these processes still remain elusive. We used a mouse strain with mutant NF-kappaB inducing kinase (NIK) to examine the mechanisms underlying the breakdown of self-tolerance. This NIK-mutant strain manifests autoimmunity and disorganized thymic structure with abnormal expression of Rel proteins in the stroma. Production of immunoregulatory T cells that control autoreactive T cells was impaired in NIK-mutant mice. The autoimmune disease seen in NIK-mutant mice was reproduced in athymic nude mice by grafting embryonic thymus from NIK-mutant mice, and this was rescued by supply of exogenous immunoregulatory T cells. Impaired production of immunoregulatory T cells by thymic stroma without normal NIK was associated with altered expression of peripheral tissue-restricted Ags, suggesting an essential role of NIK in the thymic microenvironment in the establishment of central tolerance.

子宮内胎仔操作法による細胞移植

羊膜上皮細胞の肝細胞様分化と再生医療への応用可能性について。

NF-kappaB-inducing kinase (NIK) is involved in lymphoid organogenesis in mice through lymphotoxin-beta receptor signaling. To clarify the roles of NIK in T cell activation through TCR/CD3 and costimulation pathways, we have studied the function of T cells from aly mice, a strain with mutant NIK. NIK mutant T cells showed impaired proliferation and IL-2 production in response to anti-CD3 stimulation, and these effects were caused by impaired NF-kappaB activity in both mature and immature T cells; the impaired NF-kappaB activity in mature T cells was also associated with the failure of maintenance of activated NF-kappaB. In contrast, responses to costimulatory signals were largely retained in aly mice, suggesting that NIK is not uniquely coupled to the costimulatory pathways. When NIK mutant T cells were stimulated in the presence of a protein kinase C (PKC) inhibitor, proliferative responses were abrogated more severely than in control mice, suggesting that both NIK and PKC control T cell activation in a cooperative manner. We also demonstrated that NIK and PKC are involved in distinct NF-kappaB activation pathways downstream of TCR/CD3. These results suggest critical roles for NIK in setting the threshold for T cell activation, and partly account for the immunodeficiency in aly mice.

It has been hoped that amniotic epithelial cells would be a gene carrier to neural and hepatic tissue, because of 1) the presence of neural and hepatic stem-like cells, 2) the ability to cryopreserve them, 3) long-term survival in the transplanted site, and 4) few ethical problems concerning procurement. But transplantation of a sufficient number of cells to adult tissue needs large-scale cell supply and may lead to vascular embolism. We attempted transplantation of amniotic epithelial cells into fetal liver, because 1) the fetal liver is at the proliferative stage, 2) the number of cells required is small, and 3) the fetal stage is advantageous for the induction of immunological tolerance. Amniotic epithelial cells from day 18.5-20.5 fetuses were transfected with adenoviral AdlacZ and harvested to inject into fetal rat liver of the syngeneic strain (day 18.5-20.5). The efficacy of cell transplantation into the liver increased in the order: intraplacental < intraumbilical vein < intrahepatic route. LacZ-transfected amniotic cells (1-8 x 10(5) cells), hepatocytes (5 x 10(5) cells), or AdlacZ vector solution (1.7 x 10(7) pfu) were injected through the uterine membrane into the liver. Transplanted cells formed a cellular mass and survived for up to 14 days after birth, whereas lacZ-transfected cells were rapidly decreased after the injection of AdlacZ vector or rat hepatocytes as a gene carrier so that the use of amniotic epithelial cells as a gene carrier will result in long-term expression of exogenous genes in the liver.

It is hoped that amniotic epithelial cells can be useful in cell-mediated gene therapy. We report here an experimental cell transplantation model of amniotic cells in rats. There is an anatomical difference between human and rodent embryos. We established a method to isolate amniotic cells that are equivalent to human amniotic epithelial cells. An amniotic membrane distinct from the yolk sac was carefully collected and teased in saline containing deoxyribonuclease and hyaluronidase, followed by collagenase digestion. The cell yield was approximately 10(6) cells per pregnant female (10(5) cells per fetus), roughly in proportion to the age of fetus used, and 60% of the isolated cells were attached to the dish under culture conditions. Telomerase activity was higher in the cells isolated from fetuses in the middle stage (day 13.5 to 15.5) than in the late stage (day 17.5 to 21.5). Adherent cells exhibited two to three times more cell division, resulting in a ninefold increase in the number of cells. Immunohistochemical analysis revealed that approximately half of the adherent cells were albumin positive and formed clusters. The senescent cells survived for 2 months without apparent morphological changes. The adherent cells were able to be stored in liquid nitrogen and had a viability of 70% when thawed. Gene transduction with adenovirus vector was highly effective for rat amniotic cells, Transplantation of lacZ transfected amniotic cells into syngeneic rat liver resulted in the integration of the transplanted cells in the liver structure and the cells survived for at least 30 days.

Alymphoplasia (aly) mice, a natural strain with a mutant NF-kappa B-inducing kinase (NIK) gene, manifest a unique phenotype; they lack lymph nodes and Peyer's patches, have a disturbed spleen architecture, and exhibit defects in both Ab and cellular immune responses. Although a stromal defect caused by impaired lymphotoxin-beta receptor signaling accounts for their abnormal lymphoid organogenesis, the exact mechanisms underlying the development of immunodeficiency in aly mice are poorly understood. We therefore investigated the contribution of hemopoietic cells with the aly NIK mutation to the development of immunodeficiency. Transfer of aly/aly bone marrow cells into aly/+ mice resulted in poorly developed B cell follicles and lack of support for the development of germinal centers and isotype switching, indicating that the hemopoietic cells of aly mice contain an autonomous defect. However, follicular dendritic cell clusters were maintained in the spleens of these bone marrow chimeras, suggesting that the lack of follicular dendritic cell clusters in aly mice is probably due to the stromal defect, The aly mice larked marginal zone B cells in their spleens, and aly/aly B cells showed an impaired proliferative response after in vitro stimulation. IL-2 production by activated T cells was also impaired. By contrast, the dendritic cells of aly mice exhibited grossly normal development and function. Supporting the concept of an autonomous cell defect, Rel protein expression was altered in aly/aly spleens. Thus, the aly NIK mutation affects hemopoietic cell function in an intrinsic fashion and, together with the stromal defect, may contribute to the development of immunodeficiency in aly mice.

Single blastomeres from eight-cell stage bovine embryos matured and fertilized in vitro were electrically fused with enucleated oocytes matured in vitro. In experiment-1, The percentage of these reconstituted embryos developed to the two- to eight-cell stage 48 hr after electrofusion was increased when both the eight-cell embryos and the enucleated oocytes were derived from oocytes cultured with granulosa cells (14% vs. 38%). In experiment 2, the relationship between activation of oocytes and developmental ability of reconstituted embryos was examined. Although both ethanol and electrical stimulation efficiently induced parthenogenetic activation of oocytes matured in vitro for 26-28 hr (ethanol, 89%; electrical stimulation, 73%), the ratio of the second polar body extrusion differed (80% vs. 22%). Ethanol-treated enucleated oocytes, however, were not significantly different from the early-cleavage of the reconstituted embryos 48 hr after electrofusion (nontreated, 38%; treated, 43%). In experiment 3, reconstituted embryos at the two- to eight-cell stage 48 hr after the electrofusion were cocultured with granulosa cells for 6-7 days. Of 69 embryos, one developed to a morula and three developed to blastocysts.

The spiny rat (genus Tokudaia), which has XO-type sex chromosomes without Y-chromosomes, inhabits only in Japan. Although this species is endangered and therefore difficult to utilize in fundamental research, we have attempted to analyze the sex chromosome composition of mature mammalian spermatozoa with XO-type chromosome configuration to provide evidence for a direct discussion of these meiotic mechanisms. However, as a preliminary step to approach rare samples, we have attempted to isolate sperm using FACS and segregate X- and Y-sperm by FISH in several model rodents, but without significant results in either case, we are continuing our research. On the other hand, we have obtained some secondary information that contributes to the reproductive engineering approach for wild-derived rodents.

In this project, we showed that artificial nuclear F-actin associates with chromatin and that nuclear F-actin affects Wnt/beta-catenin signaling, which has important roles on gene reprogramming and cell development. Indeed, the expression of OCT4 is shown to be regulated by nuclear F-actin. By screening a peptide library, we identified bicyclic peptides binding to G- or F-actin. We also showed a possibility that these bicyclic peptides are useful for artificial regulation of nuclear F-actin.

During the maternal-to-zygotic transition (MZT), maternal mRNAs and proteins are degraded and numerous genes are zygotically activated. We have previously shown that the transient inhibition of ubiquitin-proteasome system (UPS) after fertilization leads to a delay in the initiation of zygotic transcription and impaired full-term development in mouse. These results suggest that the degradation of maternal proteins by UPS during MZT is crucial for developmental reprogramming. However, the maternal proteins, which are degraded by UPS during MZT and can contribute to the zygotic transcription and normal development, have not been identified. In this study, we identified such maternal proteins, which is involved in the SUMO modification, and screened several candidates by examining the effects of their overexpression on preimplantation development of mouse embryos. These results suggests that a nuclear SUMO ligase is a candidate as maternal proteins degraded by UPS during MZT.

We induced the formation of nuclear actin filaments in cultured cells, and anlyzed gene transcription. In the cells, the expression of multiple genes, including OCT4, were changed. We then analyzed the effect of nuclear actin filaments on Wnt/beta-catenin signaling. We found that nuclear actin filaments induced the accumulation of beta-catenin in the nucleus and activate targets genes. In addition, nuclear actin filaments associated the promoter of the genes.

In this study from the viewpoint of spatial positioning of chromosome territories and genomic regions within the nucleus I would try to find the molecular mechanism of why the iPS cells are established and to investigate gene kissing of reprograming related genes by 3D-FISH techniques using the DNA probes with Oct3/4, Sox2, Klf4, c-Myc, and Nanog, and their chromosomal paints onto mice cell nuclei. Basic characteristics of spatial arrangements have not yet been carried out due to shortage of available cells, however, when overcoming technical problems, I would perform the analyses and discuss the issues.

Conjugated linoleic acid has been reported to be effective for cancers and obesity. In this study, a gene for an isomerase for conjugated linoleic acid (PAISOM) was introduced into bovine cells. We obtained cells stably transfected with PAISOM gene. We also successfully produced cloned embryos from the PAISOM-cells. However, Gas chromatography of fatty acids in the PAISOM-cells did not indicate the conjugated linoleic acid, but we detected a peak unknown but likely to be conjugated arachidonic acid that were elongated from conjugated linoleic acid.

本研究は、ゲノムを細胞核に機能的に折り畳むための遺伝情報を解明することを最終目標にしている。そして、本年度は、計画に沿って3つの解析を行った。以下に成果を報告する。 1. DNA高次構造のクロマチン制御能と遺伝子発現制御能の解析 : 一過的遺伝子発現系を用いた解析で、我々は既に遺伝子発現を活性化できる十字架構造とZ型DNA構造を見出していた。そこで本年度は、両者のクロマチン制御能の解析を中心とした解析計画を立てていた。しかし、再度検討した結果、この計画を実施するためには、まだ両構造の転写活性化能が不十分であることが判明した(実験的に検出できないほど僅かなクロマチン構造の変化でも転写活性に影響することがあるため)。さらに、この点を解決するためには、まずZ型DNA構造に的を絞り、転写活性化能を上昇させた上で次の解析に進むことが賢明であると判断された。そこで、新規のZ型構造の開発を引き続き行うこととし、これまでに転写を約25倍活性化できる構造を作製することに成功した。この他、マウスES細胞に導入した人工ベントDNAT20は、同細胞を肝細胞に分化させても転写を活性化する能力を保持していることを明らかにした。 2. 各種真核生物ゲノムの柔軟性地図の作成 : ヒト、チンパンジー、マウス、ショウジョウバエ、線虫、酵母、シロイヌナズナの各ゲノムの柔軟性を3bpまたは4bp単位で解析し、各ゲノムの全長に渡るDNAの柔軟性地図を完成させた。そして、どのゲノム上にも極めて柔軟な特性をもつ小領域(SPIKEと命名)が分布していることを発見した。 3. ヌクレオソームの自己集合能の解析 : ニワトリの赤血球から精製したコアヒストンと各種DNAを用いて試験管内でヌクレオソームを再構成した。得られたヌクレオソームの混合溶液を解析した結果、同じDNAをもつヌクレオソーム同士が集合する傾向があることが示唆された。

The T20 is a synthetic 180 by bent DNA fragment that mimics a part of left-handed supercoils. T20 can activate transcription by organizing local chromatin structure in HeLa cells. This study investigated whether T20 could activate transcription in the genome of mouse ES cells. T20 was found to activate the adjacent promoter even in the ES cells. Furthermore, the function of T20 was maintained even when the cells were differentiated into hepatocytes. We also investigated the locus of the reporter by using lacO/lacI-GFP transgene detection system. We constructed a tandem-repeat of the lac operator sequences(64 repeats), ligated it to the reporter construct, and integrated the resulting construct into the genome of ES cells. The T20-containing reporter was found located in the periphery of the nucleus. II. Genetic information carried in physical properties of DNA We made a program that can calculate the flexibility profile of a given DNA. Using it, we investigated the flexibility profiles of all of the recombination hot spots of Saccharomyces cerevisiae chromosome 3. These sites were indicated to be slightly more flexible when compared to the average flexibility of the yeast genome. In addition, we found that the exons are more flexible than the introns. This difference may function as a signal for some proteins to distinguish these two regions.

T20(負の超らせんを擬態した180bpの人工ベントDNA)はクロマチン内での転写を活性化できる。平成18年度は、ゲノムにT20を保有するマウスのES細胞株を用いて、T20が細胞の分化に及ぼす影響について調べた。この細胞株は分化誘導の第1段階(中・内胚葉系細胞への分化誘導)では特に異常を示さなかったが、分化誘導の第2段階(肝細胞への分化誘導)に移行させる際に、条件によっては細胞増殖が停止してしまうことが明らかになった。なお、T20をもたない対照株では、問題なく肝細胞に分化した。この結果は、T20が細胞の分化機構にも関与しうることを示唆しており、興味深い発見と思われる。また、T20をもつ細胞株は、対照株にくらべて増殖速度が約1.5倍速いことが明らかになり、T20はDNA複製にも影響を及ぼす構造であることが示唆された。この他、平成18年度は、DNAの柔軟・剛直特性をゲノムワイドに解析できるコンピュータプログラムの開発を試みた。その結果、300MbのDNAの機械的特性をわずか1時間程度で解析できるプログラムを作成することに成功した。このプログラムを用いた解析は緒についたばかりであるが、興味深い知見が得られ始めている。たとえば、酵母第3染色体を用いた解析では、遺伝子間領域(プロモーターを含む)は、ORF (open reading frame)よりも硬いDNAでできていることが判明した。さらに当該年度は、二本鎖DNAに自己集合の能力があることと、DNAの自己集合は、生理的濃度のマグネシウムイオンの存在下で顕著に起こり、電気泳動でも分離しないほど安定であることを論文で発表した。

The purpose of this study is to produce cattle containing healthy polyunsaturated fatty acids that are not biosynthesized by mammals. To achieve this purpose, we developed a method to produce transgenic cattle carrying a gene for a fatty acid desaturase taken from plants that are able to newly biosynthesize the polyunsaturated fatty acids. We obtained an FAD3 cDNA which encoded an ω3 fatty acid desaturase from flax seeds that contained the highest rate of α-linolenic acid (18:3n-3) contents in land plants. New synthesis of 18:3n-3 was observed when the cDNA was introduced into yeast and examined their fatty acid composition. Furthermore, a transfection method of a foreign gene stably into mammalian primary cultured cell was established. Using this method, pβ-act/FAD3/IRES/EGFP(neor) was transfected into bovine fibroblast cells and mouse 3T3-L1 cells, and then the functional expression was examined by determination of composition rates of 18:3n-3. The FAD3 gene derived from flax seeds expressed functionally in mammalian cultured cells, because the ratio of the 18:3n-3 increased in the transfected cells with the FAD3 gene compared with that in wild-type cells. However, the ratio in the transfected cells were low and only 0.4% (wild type : 0.2%). Therefore, the DNA sequence of the FAD3 that was a plant type was changed to the mammalian-type DNA sequence according to the mammalian codon usage. Then we obtained a new FAD3 gene optimized in mammalian cells (FAD3opt), and expression vector (pCAG/FAD3opt/IRES/EGFP(neor)) was produced. Strong expression of EGFP was observed in the mouse 3T3-L1 cells transfected with pCAG/FAD3opt/IRES/EGFP(neor). In addition, we have obtained a bovine primary fibroblast cell lines carrying pCAG/FAD3opt/IRES/EGFP(neor). We now examine the fatty acid composition in the transfected cells. We also examined the relation of gene expression in bovine cloned embryos to their further development, to establish an effective method for production of cloned calves with the transgenic cells as donor cells. We examined gene expression in embryos reconstructed with bovine fibroblasts carrying a luciferase expression vector (pβ-act/luc+/IRES/EGFP(neor)). As a result, when intensity of LUC+ expression was examined in transgenic cloned embryos 60 hours post fusion (hpf), strongly expressed-embryos developed at a high rate. Moreover, when The embryos were then classified as being Luc-positive, mosaic, or Luc-negative depending on whether all, some or no blastomeres were luminescent, respectively, the developmental rates to the blastocyst stage of positive embryos were higher than those of mosaic embryos. Any negative embryos did not developed to blastocysts. Then, we examined DNA methylation levels in the embryos by immunostaining with a 5-methyl cytosine antibody. The methylation level of the Luc-positive embryos was lower than that of the Luc-negative embryos. These results indicated that the developmental capacity of NT embryos could be correlated positively to the gene expression ability and negatively to the DNA methylation level at 4- to 8-cell stage.

ES細胞に未発現のターゲット遺伝子のへshRNAの導入の第1段階としてハイグロマイシン耐性フィーダーを用いて、ターゲットの直鎖状化したHAタグ遺伝子を融合させた未発現遺伝子であるG93ASOD1変異遺伝子を発現ベクターに組み込み、これをエレクトロポレーションした。ハイグロマイシンで耐性ES細胞を選択して、50個の耐性ES細胞クローンの中からHAタグでウエスタンブロットを行いターゲット遺伝子を安定を発現したES細胞クローンが得られた。 第2段階として未発現遺伝子が安定発現したES細胞ラインに対して、直鎖状化したShort-hairpinタイプのsiRNA発現ベクターをエレクトロポレーションしてネオマイシンでshRNAを安定発現ES細胞をクローン化した。HAタグタグでウエスタンブロットを行いターゲットG93ASOD1遺伝子発現が抑制されているG93ASOD1変異遺伝子とshRNAの両者が安定発現し、第1段階で発現させたG93ASOD1を有効に抑制しているES細胞クローンが作製できた。 現在このダブル遺伝子安定発現ES細胞クローンをブラストシストにマイクロインジェクションしてキメラマウスを複数のクローンで作製したが、ESの寄与率が悪く、トラブルシュートとして第1段階のES細胞クローンでキメラマウスを作製中である。

哺乳動物の遺伝子機能の解析において、遺伝子ターゲティングによるノックアウトマウス作製技術は個体レベルでの解析システムとして、既に必須のツールとなっている。しかしながら、現行のノックアウトマウスの作製過程では胚性幹細胞(ES細胞)に遺伝子改変操作を行い、その後、キメラマウスの交配によってはじめてへテロマウスが樹立されるため、ホモ個体の樹立までには、長い時間を要する。しかしながら、もし仮に遺伝子改変操作を行った細胞から直接へテロマウスを作製することができれば、研究効率を著しく改善できることは論を特たない。このような視点から、最近報告された体細胞を用いたクローンマウス作製技術を応用し、遺伝子改変操作を行なった体細胞の核移植操作により、直接へテロマウスを取得する系の開発を以下のごとく試みた。 1)ターゲティングベクターを導入する体細胞の選択 過去に体細胞での相同遺伝子組換え効率はES細胞に比べ低いことが報告されたが、近年、その改善が著しいベクターや遺伝子導入方法用いた相同組換え効率については再検討の余地がある。そこで既存のベクターを用いて胎仔線維芽細胞への遺伝子導入を行なった。胎仔線維芽細胞は、遺伝子導入の効率については優れていたものの、相同組換え体を取得するには至らなかった。 2)体細胞に由来する個体作製技術の確立 上記と同じく、胎仔線維芽細胞の核移植により体細胞クローンの作出を試みたが、今回の研究では技術的に、依然、きわめて困難であった。 以上、本年度の研究からは体細胞クローン技術を応用した簡便なノックアウトマウス作製方法を確立するには至らなかったが、理論的にも十分に可能性がおり、さらに研究を進める必要がある。

リンホトキシンノックアウトマウスを用いた研究から、私達はリンホトキシンが濾胞樹状細胞(FDC)の構築を誘導する作用をもったユニークなサイトカインであることを明らかにしてきた。しかしながら、リンホトキシン非存在下でFDCがどのような存在様式をとっているか、あるいはFDC前駆細胞がリンホトキシンのシグナルを受けた時、一体どのようにしてFDC clusterという高次構築をとるかという点が不明である。この問題を研究するため、FDCを蛍光物質GFPにより標識し、リンホトキシン受容体を介するシグナル伝達を遮断した場合にFDCの消長を追跡できる実験系の開発を試みた。すなわち、リンホトキシンのシグナル伝達が生体内でのFDC cluster構築をどのようにコントロールするかについての研究を以下の方法により計画した。 1)FDCで強く発現されている補体レセプターの遺伝子プロモーターにGFPを連結した導入遺伝子を構築し、トランスジェニックマウスを作製する。 2)B細胞からのGFP発現を消去するため、正常マウスからの骨髄移植を併用し、FDC特異的にGFPを発現するトランスジェニックマウスを樹立する。 3)このようなマウスにリンホトキシン受容体-IgG融合蛋白を投与してリンホトキシンの作用を遮断し、経時的にマウス脾臓のGFP発現様式を観察して、FDCの消長をモニターする。 現在、トランスジェニックマウスの樹立作業を行なっており、目的とするマウスの取得とともに、2)以下の解析実験に着手したい。 FDCは液性免疫の成立に重要なはたらきをもつが、同時にHIVウイルスのreservoirとしての役割も指摘されており、FDCがリンホトキシンに依存して存在形態を変化させるメカニズムを詳細に解析することは、免疫システムの構築機構を明らかにするのみならず、臨床免疫学の観点からも意義深いものと思われる。

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