Development of efficient gene disruption system in any tissues or organs

在任何组织或器官中开发有效的基因破坏系统

• 批准号: 08557065
• 负责人: TAKEDA Junji
• 金额: $ 10.3万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08557065/
• 关键词: Cre; loxp; Hematopoietic stem cell; Tissue specific gene targeting; Pig-a; Pig-a; コンディショナルジーンターデッティング; 膜融合リポソーム; コンディショナルジーンターゲティング

Gene disruption in murine embryonic stem (ES) cells allows to study functions of endogenous genes after establishing mice derived from targeted ES cells. Since many gene disruptions cause embryonic lethality, it is not possible to analyze specific gene functions in adult mice. To circumvent these difficulties, we applied Cre/loxP system. The system allows to disrupt a gene flanked by identically oriented loxP sites with Cre-expression-dependent manner.In this study, we tried to apply two methods to introduce Cre recombinase into mouse genome, exogenous and endogenous way. For the former, liposome containing Gre was produced. Addition of the liposome to hematopoietic cells in vitro resulted in disruption of loxP containing gene (Pig-a). However, transfer of these cells into lethally irradiated mice revealed that Pig-a disruption did not occur in the hematopoietic stem cells. This result suggest that the exogenous Gre by the liposome has limitation to introduce Cre into the genome. For the latter, we established a series of Gre transgenic mice. Cre transgene worked very efficiently including hematopoietic stem cells.

在小鼠胚胎干细胞（ES细胞）中进行基因破坏允许在建立源自靶向ES细胞的小鼠后研究内源基因的功能。由于许多基因破坏导致胚胎死亡，因此不可能分析成年小鼠的特定基因功能。为了克服这些困难，我们采用了Cre/loxP系统。本研究尝试采用外源和内源两种方法将Cre重组酶导入小鼠基因组中。对于前者，制备了含有Gre的脂质体。在体外向造血细胞中加入脂质体导致含有loxP的基因（Pig-a）的破坏。然而，将这些细胞转移到致死性照射的小鼠中揭示了在造血干细胞中没有发生Pig-a破坏。这一结果表明，脂质体介导外源Cre基因导入基因组存在一定的局限性。对于后者，我们建立了一系列的Gre转基因小鼠。Cre转基因在造血干细胞中的作用非常有效。

项目成果

Takahama,Y.: "Functional competence of T cells in the absence of GP1-anchored proteins caused by T-cell specific disruption of Pig-a gene." Eur.J.Immunol.28. 2159-2166 (1998)

Takahama,Y.：“在缺乏 GP1 锚定蛋白的情况下，T 细胞的功能能力是由 T 细胞特异性破坏 Pig-a 基因引起的。”

Nozaki, M., et al.: "Developmental abnormalities of glycosylphosphatidylinositol-anchor deficient embryos revealed by Cre/loxP system" Lab.Invest.(in press). (1999)

Nozaki, M., et al.：“Cre/loxP 系统揭示的糖基磷脂酰肌醇锚定缺陷胚胎的发育异常”Lab.Invest.（出版中）。

Takeda, K.et al.: "Stat3 activation is responsible for IL-6-dependent T cell proliferation through preventing characteraization of T cell specific stat3-deficient-mice." J.Immunol. 161. 4652-4660 (1998)

Takeda, K.等人：“Stat3 激活通过阻止 T 细胞特异性 stat3 缺陷小鼠的表征来负责 IL-6 依赖性 T 细胞增殖。”

Takeda,K.: "Stat3 Activation is Responsible for IL-6-Dependent T Cell Proliferation Through Preventing Apoptosis : Generation and Characterization of T Cell-Specific Stat3-Deficient Mice" J.Immunol.161. 4652-4660 (1998)

Takeda,K.：“Stat3 激活通过预防细胞凋亡负责 IL-6 依赖性 T 细胞增殖：T 细胞特异性 Stat3 缺陷小鼠的生成和表征”J.Immunol.161。

Minoru Takahashiet al.: "PIG-B,a membrane protein of the endoplasmic reticulum with a large lumenal domain,is involved in transferring the third mannose of the GPI anchor." The EMBO Journal. 15・16. 4254-4261 (1996)

Minoru Takahashiet al.：“PIG-B 是一种具有大腔域的内质网膜蛋白，参与 GPI 锚定的第三个甘露糖的转移。” EMBO 杂志 15・16。