Cloning of apoptosis-inducing genes by introducing genes into living tadpole

本研究的目的是克隆两栖类变态过程中蝌蚪退化尾中甲状腺激素（TH）诱导的退化诱导基因.我们分离了非洲爪蟾Caspase家族的所有基因，并将其导入尾源性成肌细胞系。具有长前结构域的Caspases的过表达显示出强的细胞杀伤活性。爪蟾Caspase家族基因的表达在退化的蝌蚪尾中上调，而在TH处理的死亡的尾源细胞系中没有上调，这表明Caspase家族新的RNA合成与程序性细胞死亡有关.许多报道支持TH诱导的分泌性细胞外基质降解酶的增加导致肌腱连接的破坏，从而使肌细胞与细胞外基质分离并导致其细胞死亡的谋杀模型。然而，当TH信号被显性蛋白的强制表达阻断时， ...更多信息 T-阴性甲状腺激素受体（DNTR），TH处理的成肌细胞系的死亡被抑制，表明程序性细胞死亡通过自杀机制发生。为了证实这一点，将DNTR表达构建体与标记基因一起注射到活蝌蚪的尾肌细胞中，并在尾部完全吸收之前检测标记蛋白的活性。仅在与DNTR表达构建体共注射的尾部中观察到肌细胞中的标记基因表达。这一结果表明，肌细胞的死亡是对TH的一种自杀反应.利用体内电穿孔将标记基因和cDNA文库导入表达载体中的蝌蚪尾，几天后切下用于器官培养，并检查标记基因的表达。没有分离出任何克隆作为抑制标记基因表达的克隆。到目前为止，通过在退化尾中增加表达而鉴定的几个基因的过表达也没有显示出任何抑制活性。我们正在分析一组cDNA克隆，这些克隆在尾源性成膜细胞系的转染筛选中对标记基因表达具有显著抑制活性。少

Our purpose is to clone apoptosis-inducing genes that are induced by thyroid hormone (TH) in the regressing tail of tadpole during amphibian metamorphosis.1. We have isolated all Xenopus Caspase family genes as apoptosis-excuting genes, and introduced them into tail-derived myoblast cell line. The overexpression of Caspases with a long prodomain showed a strong cell-killing activity. The expression of Xenopus Caspase family genes was upregulated in the regressing tail of metamorphosing tadpole, but was not in dying tail-derived cell line treated with TH, which demonstrates that new RNA synthesis of caspases is dispensable to programmed cell death.2. Many reports supported the murder model that the increase of secreted extracellular matrixdegrading enzymes induced by TH results in the disruption of the myotendinous junctions, which detaches muscle cells from the extracellular matrix and causes their cell death. However, when TH-signaling was blocked by the enforced expression of dominan … More t-negative thyroid hormone receptor (DNTR), the death of TH-treated myoblast cell line was repressed, suggesting that the programmed cell death takes place through a suicide mechanism. To confirm it, DNTR expression construct was injected into the tail muscle cells of living tadpole with a marker gene, and the activity of marker protein was detected just before tail resorbed completely. The marker gene expression in muscle cells was observed only in tails co-injected with DNTR expression construct. This result indicates that muscle cell dies by suicide as a response to TH in the regressing tail.3. Tadpole tail was introduced with a marker gene and cDNA library in a expression vector using in vivo electroporation, cut out for the organ culture several days later, and examined on the maker gene expression. Any clone was not isolated as one inhibiting marker gene expression. The overexpression of several genes identified so far by the increased expression in the regressing tail did not show any suppressive activity, either. We are analyzing a group of cDNA clones which has the significant suppressive activity to the marker gene expression in the screening by transfection of tail-derived mvoblastic cell line. Less