Identification and separation of pluripotential tissue stem cells and its application for producing somatic cell nuclear transferred cloned mouse

多能组织干细胞的鉴定分离及其在体细胞核移植克隆小鼠生产中的应用

• 批准号: 15580251
• 负责人: MITANI Tasuku
• 金额: $ 1.66万
• 依托单位: Kinki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15580251/
• 关键词: amniotic epithelial cells; hepatic function; interferon-?; nuclear transfer; somatic clone animals; embryonic stem cells; RNA interference; knockdown mouse; 精原細胞; フローサイトメトリー; 磁気細胞分離システム; α6インテグリン; RNAi; ES細胞; ALS; 組織幹細胞; 主要組織適合性抗原; 細胞移植 /

In order to develop a novel method to produce genetically-modified food animals, we aimed to separate somatic stem cells in various tissues applicable for donor cells for producing cloned mice. We also examined an establishment of ES cells from nuclear transferred embryos and their ability to differentiate. Moreover, we examined manipulating an expression of certain target gene.1.Multipotential amniotic epithelial cells (AECs) were cultured and characterized their properties. AECs showed moderate expression of MHC class I and II and the expression of those molecules by IFN-? was only slightly induced. It was notably that AECs expressed some major hepatic genes. Spermatogonial stem cells (SSC) were enriched from testes using MACS for CD9 or ?6-integrin. SSCs could be cultured in vitro for more than 2 months with expression of CD9, ?6-integrin and Oct4 genes.2.As aire-deficient mice showed insufficiency of gametogenesis, the expression of aire was examined in gonads and ES cells. AIRE proteins were partially distributed in immature thymus, ovary and ES cells and localized in the nuclei forming nuclear dots.3.AECs from EGFP-transgenic mice were examined for nuclear transfer. Using the reconstituted embryos which developed to the blastocyst stage, ES cells were established. ES cells were also established from parthenotes and were confirmed their ability to differentiate in vitro and in vivo.4.Embryonic fibroblasts, cumulus cells, ES cells and AECs were used for producing cloned mouse. Cloned mouse derived from cumulus cells was produced. Factors affecting developmental ability of reconstituted embryos and conditions for improvement of their development were investigated.5.RNAi-expression vector for SOD1, mutant protein of which induces ALS, was introduced into ES cells and using its stable transfectant, knockdown mice for SOD1 were produced. By crossing this SOD1-siRNA transgenic mouse with human ALS model mouse, siRNA prevented the development ALS.

为了开发一种生产转基因食用动物的新方法，我们的目标是从不同组织中分离适合于供体细胞的体细胞，以生产克隆小鼠。我们还检测了核移植胚胎中ES细胞的建立及其分化能力。此外，我们还研究了特定靶基因的表达。1.培养多潜能羊膜上皮细胞(AEC)并对其特性进行鉴定。血管内皮细胞呈中等水平表达MHC-I、II类分子，且在干扰素？只是轻微的诱导。值得注意的是，AECs表达了一些主要的肝脏基因。精原干细胞(SSC)用巨噬细胞集落刺激因子(MACs)进行CD9或β6整合素的培养。SSCs可在体外培养2个月以上，并表达CD9、β6整合素和Oct4基因。2.针对AIRE基因缺陷小鼠配子发生不足的情况，检测AIRE基因在性腺和ES细胞中的表达。AIRE蛋白部分分布于未成熟的胸腺、卵巢和ES细胞中，并定位于形成核点的核中。3.检测转EGFP基因小鼠AEC的核移植情况。利用发育到囊胚期的重组胚胎建立ES细胞。胚胎成纤维细胞、卵丘细胞、胚胎干细胞和血管内皮细胞用于制备克隆小鼠。获得了来源于卵丘细胞的克隆小鼠。研究了影响重组胚胎发育能力的因素及改善重组胚胎发育的条件。5.将突变蛋白SOD1RNAi表达载体导入ES细胞，利用其稳定的转染体获得SOD1RNAi表达载体，获得SOD1RNAi敲除小鼠。通过将SOD1-siRNA转基因小鼠与人ALS模型小鼠杂交，siRNA阻止了ALS的发生。

项目成果

マウスautoimmune regulator (aire)タンパク質の局在に関する組織学的解析。

小鼠自身免疫调节蛋白 (aire) 定位的组织学分析。

• 发表时间: 2006
• 期刊: Mem. Inst. Adv. Technol. Kinki Univ. 11
• 作者: T.Mitani;H.Kato;増田淳大
• 通讯作者: 増田淳大

Expression of zag1 in mouse preimplantation embryos.

Zag1 在小鼠植入前胚胎中的表达。

• 发表时间: 2005
• 期刊: Reprod.Fert.Dev 17
• 作者: A.Kasamatsu;K.Saeki et al.;K.Matsumoto et al.
• 通讯作者: K.Matsumoto et al.

NF-kappa B-inducing kinase establishes self-tolerance in a thymic stroma-dependent manner.

• 发表时间: 2004
• 期刊: Journal of immunology
• 影响因子: 4.4
• 作者: Fumiko Kajiura;Shijie Sun;T. Nomura;K. Izumi;T. Ueno;Y. Bando;N. Kuroda;Hongwei Han;Yi Li

In vitro culture of CD9-expressing cells enriched by magnetic cell sorting from testes of cryptorchid adult and pup in mice

通过磁性细胞分选富集小鼠隐睾成虫和幼崽睾丸的 CD9 表达细胞的体外培养

• 发表时间: 2006
• 期刊: Reprod.Fert.Dev. 18
• 作者: Tsuji;I;T.Mitani et al.
• 通讯作者: T.Mitani et al.

Cytological analysis of hepatic gene expression and immunological response of MHC antigens in mouse amniotic epithelial cells.

小鼠羊膜上皮细胞中 MHC 抗原的肝基因表达和免疫反应的细胞学分析。

• 发表时间: 2005
• 期刊: Reprod.Fertil.Dev. 17
• 作者: Mitani;T
• 通讯作者: T