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Study for the cell lineage and tissue specificity in embryonic cells using transgenic Xenopus frogs

使用转基因爪蟾蛙胚胎细胞的细胞谱系和组织特异性研究

基本信息

批准号：
17570176
负责人：
SAKAMAKI Kazuhiro
金额：
$ 2.3万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17570176/
关键词：
Transgenic Xenopus fluorescent protein Cre loxP

项目摘要

Fate maps of embryonic cells or tissues during morphogenesisare useful for understanding the development. To urge fate-mapping studies, we aimed to establish a Cre/loxP system combined with transgenesis in Xenopus frogs. Because it was expected that the Cre/loxP system with transgenic frogs shows the advantage for tracing cells or tissues over a long period. We constructed the transgene, CAG promoter-loxP/EGFP/IoxP-DsRed2 (tentatively named as EGFP/DsRed) and inserted it into fertilized eggs by microinjection. The established transgenic Xenopus line (Green/Red-Tg) ubiquitously expressed EGFP in the whole body. Furthermore, by intercrossing this line with another transgenic line expressing Cre recombinase under the control of the muscle-specific cardiac actin promoter, we found that DsRed2 fluorescent proteins were expressed in embryos in a muscle-specific manner (Waldner et al., Dev. Dyn., 2006). Thus, we showed that the Cre/loxP system works in amphibians as well as mammals, and our Green/Red Tg frogs are useful as a reporter animal. We also developed a technique by electroporationforintroducingCremRNAintoembryos.Usingourtransgenicfrogs, we are now studying how left-right (LR) positional information inducing anatomical asymmetry is determined during embryogenesis.

胚胎细胞或组织在形态发生过程中的命运图对于理解发育是有用的。为了促进非洲爪蟾命运定位研究，我们旨在建立一种结合转基因的Cre/loxP系统。因为人们预计转基因青蛙的Cre/loxP系统在长期追踪细胞或组织方面具有优势。我们构建了转基因CAG启动子loxp /EGFP/IoxP-DsRed2（暂命名为EGFP/DsRed），并通过显微注射将其插入受精卵中。建立的转基因非洲爪蟾株系（Green/Red-Tg）在全身普遍表达EGFP。此外，通过在肌肉特异性心脏肌动蛋白启动子的控制下，将该品系与另一个表达Cre重组酶的转基因品系杂交，我们发现DsRed2荧光蛋白在胚胎中以肌肉特异性方式表达（Waldner et al., Dev. Dyn., 2006）。因此，我们证明了Cre/loxP系统在两栖动物和哺乳动物中都有效，我们的绿色/红色Tg青蛙是有用的报告动物。我们还开发了一种通过电穿孔将cremrna导入胚胎的技术。利用我们的转基因青蛙，我们现在正在研究在胚胎发生过程中如何确定诱导解剖不对称的左右（LR）位置信息。