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有助于理解胚胎的发育。为了促进命运图谱的研究，我们的目标是在非洲爪蛙中建立一个结合转基因的Cre/loxP系统。因为预期转基因青蛙的Cre/loxP系统在长期示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(暂定为EGFP/DsRed)，并将其显微注射入受精卵。所建立的转基因非洲爪哇品系(Green/Red-TG)在全身均能表达绿色荧光蛋白。此外，通过将该品系与另一个在肌肉特异的心脏肌动蛋白启动子控制下表达Cre重组酶的转基因株杂交，我们发现DsRed2荧光蛋白以肌肉特异的方式在胚胎中表达(Waldner等人，Dev。Dyn.，2006)。因此，我们表明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青蛙作为报告动物是有用的。我们还开发了一项由electroporationforintroducingCremRNAintoembryos.Usingourtransgenicfrogs，开发的技术，我们现在正在研究导致解剖不对称的左右位置信息是如何在胚胎发育过程中确定的。

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.