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

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。为了促进命运定位研究，我们的目的是建立一个Cre/loxP系统结合转基因非洲爪蟾蛙。因为预期使用转基因青蛙的Cre/loxP系统在长期追踪细胞或组织方面显示出优势。构建了CAG启动子-loxP/EGFP/IoxP-DsRed 2（暂命名为EGFP/DsRed 2）转基因载体，并通过显微注射法将其插入受精卵中。建立的转基因非洲爪蟾品系（绿色/红色-Tg）在整个身体中普遍表达EGFP。此外，通过将该品系与另一个在肌肉特异性心脏肌动蛋白启动子控制下表达Cre重组酶的转基因品系杂交，我们发现DsRed 2荧光蛋白以肌肉特异性方式在胚胎中表达（Walkman等人，Dev. Dyn.，2006年）。因此，我们表明Cre/loxP系统在两栖动物和哺乳动物中起作用，并且我们的绿色/红色Tg青蛙可用作报告动物。我们还开发了一种通过电穿孔将CremRNA导入胚胎的技术。使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诱导解剖不对称的左右（LR）位置信息是如何确定的。

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.