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

为了开发一种生产转基因食用动物的新方法，我们的目标是在各种组织中分离体细胞干细胞，用于生产克隆小鼠的供体细胞。我们还研究了从核移植胚胎中建立胚胎干细胞及其分化能力。此外，我们还研究了操纵某些目标基因的表达。培养多电位羊膜上皮细胞，并对其性质进行表征。AECs显示MHC I类和II类的中等表达以及IFN-？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。用CD9或6-整合素的MACS富集睾丸精原干细胞（SSC）。ssc可在体外培养2个月以上，表达CD9、？6-整合素和Oct4基因。由于空气缺乏小鼠配子发生不足，我们在性腺和胚胎干细胞中检测了aire的表达。AIRE蛋白部分分布于未成熟胸腺、卵巢和胚胎干细胞中，并定位于细胞核内形成核点。对egfp转基因小鼠的aec进行核移植检测。利用发育至囊胚期的重组胚胎建立胚胎干细胞。从孤雌体中也建立了胚胎干细胞，并证实了其体外和体内分化的能力。胚胎成纤维细胞、胚胎积云细胞、胚胎干细胞和aec用于克隆小鼠。从积云细胞中获得克隆小鼠。探讨影响重组胚胎发育能力的因素及改善其发育的条件。将诱导ALS的突变蛋白SOD1的rnai表达载体导入ES细胞，利用其稳定的转染物，产生SOD1的敲低小鼠。通过将SOD1-siRNA转基因小鼠与人ALS模型小鼠杂交，siRNA可阻止ALS的发生。

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.