Generation of transgenic mouse expressing tamoxifen-inducible Cre recombinase

表达他莫昔芬诱导型 Cre 重组酶的转基因小鼠的产生

• 批准号: 15500297
• 负责人: ICHISE Hirotake
• 金额: $ 1.22万
• 依托单位: The University of Tokyo
• 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 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15500297/
• 关键词: tamoxifen; Cre; loxP system; FLP; FRT system; transgenic mouse; Creリコンビナーゼ

Genetic modification with Cre/loxP system in mice is widely used for the generation of tissue-specific knockouts and transgenics. However, tissue-specific genetic modification does not always rescue the mouse from unexpected embryonic death. In such cases, temporal regulation of Cre/loxP recombination is needed for Cre-expressing transgenic mice.In our research, we used two types of tamoxifen-inducible Cre recombinase, MerCreMer and CreMer (gifted by Dr.Reth, Univ.of Cologne) for the generation of Cre transgenic mice. Five transgenic mouse lines expressing MerCreMer under the control of GAG promoter (gifted by Dr.Miyazaki, Osaka Univ.) were generated with pronuclear DNA microinjection to C57BL/6J fertilized eggs. Two lines, in which MerCreMer protein expression was showed by Western blot analysis, were crossed with ROSA26R mice (purchased from Jackson laboratory). However, X-gal staining analysis of MerCreMer/ROSA26R doubly transgenic mice did not show tamoxifen-dependent cre-mediated … More excision of foxed ROSA26 gene. MerCreMer expression in the two lines was not enough to induce tamoxifen-dependent recombination.Next, we planned to generate transgenic mouse lines expressing CreMer strongly and ubiquitously. We constructed the transgene harboring GAG promoter, "frted" GFP-neo fusion gene and CreMer gene. The neo gene was derived from mutated MC1-neo-pA cassette. The transgene was electroporated into E14.1 ES cells. The embryoid bodies derived from three G418-resistant ES clones expressed GFP strongly and ubiquitously. The transient expression of FLP recombinase in the three clones induced the excision of feted GFP-neo gene and subsequent CreMer expression. ES cells before FLP/FRT recombination were introduced into C57BL/6J blastocysts. One of those clones, No.32, was germline-competent and the transgenic mouse line, CM 32, was established. Many tissues such as skeletal muscle, heart, pancreas, skin and intestine, were GFP-positive in CM32 mice. We are planning to cross CM32 mice with FLP66 mice (Takeuchi T. et al.,2002). Less

利用Cre/loxP系统对小鼠进行遗传修饰，广泛用于组织特异性基因敲除和转基因的产生。然而，组织特异性基因修饰并不总是能将小鼠从意外的胚胎死亡中拯救出来。在我们的研究中，我们使用两种类型的他莫昔芬诱导型Cre重组酶MerCreMer和CreMer（由科隆大学的Reth博士赠送）来产生Cre转基因小鼠。在GAG启动子控制下表达MerCreMer的五个转基因小鼠系（由大坂大学的宫崎博士赠送）用原核DNA显微注射法对C57 BL/6 J的受精卵进行处理。将通过蛋白质印迹分析显示MerCreMer蛋白表达的两个系与ROSA 26 R小鼠（购自杰克逊实验室）杂交。然而，MerCreMer/ROSA 26 R双转基因小鼠的X-gal染色分析没有显示他莫昔芬依赖性cre介导的细胞凋亡。 ...更多信息 切除FOXED ROSA 26基因。MerCreMer在两个品系中的表达不足以诱导他莫昔芬依赖性重组。接下来，我们计划产生强烈且普遍表达CreMer的转基因小鼠品系。我们构建了含有GAG启动子、“frted”GFP-neo融合基因和CreMer基因的转基因。neo基因来源于突变的MC 1-neo-pA盒。将转基因电穿孔到E14.1 ES细胞中。来自三个G418抗性ES克隆的胚状体强烈且普遍地表达GFP。FLP重组酶在3个克隆中的瞬时表达诱导了转染的GFP-neo基因的切除和随后的CreMer表达。将FLP/FRT重组前的ES细胞导入C57 BL/6 J囊胚。其中32号克隆具有生殖能力，建立了转基因小鼠品系CM 32。在CM 32小鼠的骨骼肌、心脏、胰腺、皮肤和肠等组织中，GFP呈阳性。我们计划将CM 32小鼠与FLP 66小鼠杂交（Takeuchi T.等，2002年）。少