Establishment of genetically modified mouse libraries using Cre/loxP gene trap

利用Cre/loxP基因陷阱建立转基因小鼠文库

• 批准号: 11558098
• 负责人: OKABE Masaru
• 金额: $ 5.82万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11558098/
• 关键词: Cre recombinase; polyA trap; ES cells; Cre; IoxP system; GFP; green mouse; ノックアウト; GFP; トランスジェニックマウス

The purpose of this study is to estimate the possibility of establishing genetically modified mouse libraries using Cre/lop trap system. The research also aimed to produce transgenic mouse lines that express Cre recombinase in various ways to enable a second generation of gene knockout.1) Gene trap in ES cells and identification of the trapped geneWe have produced trap vector that contains polyA less Puromycin resistant gene under the PGK promoter. At the same time, the vector was designed to trap a promoter of a certain endogenous gene and express Cre recombinase. Using this vector, we have obtained 600 trapped clones. After the analysis of the trapped clones using 3'-RACE, we identified 9 known gene 2 EST sequences. Other sequences were originated from unknown genes. The vector was shown to be effective to trap genes which are silent in ES cells.2) Prodction of Chimeric mouse from trapped ES cells.Chimeric mice are produced from the trapped ES cell lines and at the present more than 10 genetically modified mouse lines are established. The phenotypes of these gene-disrupted mice are now under investigations. The expression of the Cre recombinase from these trapped mice are also examined using GFP transgenic mice which becomes green in organs where Cre is expressed.In order to identify the localization of Cre expressing organs (or cells), we produced reporter "green mice" which turns fluorescent green when Cre recombinase recombine the transgene.

本研究的目的是评估利用 Cre/lop trap 系统建立转基因小鼠文库的可能性。该研究还旨在产生以多种方式表达Cre重组酶的转基因小鼠品系，以实现第二代基因敲除。1）ES细胞中的基因捕获和捕获基因的鉴定我们已经生产了在PGK启动子下含有polyA较少嘌呤霉素抗性基因的捕获载体。同时，该载体被设计为捕获某个内源基因的启动子并表达Cre重组酶。使用该载体，我们获得了 600 个捕获的克隆。使用 3'-RACE 对捕获的克隆进行分析后，我们鉴定了 9 个已知基因 2 EST 序列。其他序列源自未知基因。该载体被证明能有效捕获ES细胞中沉默的基因。2)从捕获的ES细胞中产生嵌合小鼠。嵌合小鼠是从捕获的ES细胞系中产生的，目前已建立了10多个转基因小鼠系。这些基因破坏小鼠的表型目前正在研究中。还使用 GFP 转基因小鼠检查了这些捕获小鼠的 Cre 重组酶表达，这些小鼠在表达 Cre 的器官中变成绿色。为了识别 Cre 表达器官（或细胞）的定位，我们生产了报告基因“绿色小鼠”，当 Cre 重组酶重组转基因时，它会变成荧光绿色。

项目成果

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肯，V.W.

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Harada, Y.et al.: "Postnatal Growth Failure, Short Life Span, and Early Onset of Cellular Senescence and Subsequent Immortalization in Mice Lacking the Xeroderma Pigmentosum Group G Gene"Mol Cell Biol. 19(3). 2366-72 (1999)

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