Targeted gene expression in transgenic Xenopus using the binary Gal4-UAS system
Targeted gene expression in transgenic Xenopus using the binary Gal4-UAS system
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DOI：
10.1073/pnas.022646899
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发表时间：
2002-02-05
影响因子：
11.1
通讯作者：
Amaya, E
中科院分区：
文献类型：
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作者：
Hartley, KO;Nutt, SL;Amaya, E
非洲爪蟾的转基因技术允许人们以时间和空间受控的方式错误表达基因。然而，该系统受到两个实验限制。首先，限制酶介导的整合过程依赖于染色体损伤，导致一定比例的胚胎不能正常发育。其次，每个转基因胚胎具有独特的整合位点和独特的转基因拷贝数，导致可变的转基因表达水平和可变的表型。出于这些原因，我们已经调整了Gal 4-UAS方法用于爪蟾的靶向基因表达。该技术依赖于产生携带“激活剂”或“效应子”构建体的转基因系。激活子系在所需启动子的控制下表达酵母转录因子Gal 4，而效应子系含有与目的基因连接的Gal 4-（UAS）的DNA结合基序。我们发现，在这些线的交叉，效应基因转录的时间和空间的方式激活的启动子。此外，我们使用Gal 4-UAS系统来错误表达Xvent-2，这是早期胚胎发生过程中骨形态发生蛋白4（BMP 4）信号传导的转录靶点。遗传Gal 4激活剂和Xvent-2效应转基因的胚胎显示一致的小头症表型。最后，我们利用这个系统来表征从Xvent-2的早期错误表达获得的神经和中胚层缺陷。这些结果强调了该系统在非洲爪蟾基因功能的控制分析的潜力。
The transgenic technique in Xenopus allows one to misexpress genes in a temporally and spatially controlled manner. However, this system suffers from two experimental limitations. First, the restriction enzyme-mediated integration procedure relies on chromosomal damage, resulting in a percentage of embryos failing to develop normally. Second, every transgenic embryo has unique sites of integration and unique transgene copy number, resulting in variable transgene expression levels and variable phenotypes. For these reasons, we have adapted the Gal4-UAS method for targeted gene expression to Xenopus. This technique relies on the generation of transgenic lines that carry "activator" or "effector" constructs. Activator lines express the yeast transcription factor, Gal4, under the control of a desired promoter, whereas effector lines contain DNA-binding motifs for Gal4-(UAS) linked to the gene of interest. We show that on intercrossing of these lines, the effector gene is transcribed in the temporal and spatial manner of the activator's promoter. Furthermore, we use the Gal4-UAS system to misexpress Xvent-2, a transcriptional target of bone morphogenetic protein 4 (BMP4) signaling during early embryogenesis. Embryos inheriting both the Gal4 activator and Xvent-2 effector transgenes display a consistent microcephalic phenotype. Finally, we exploit this system to characterize the neural and mesodermal defects obtained from early misexpression of Xvent-2. These results emphasize the potential of this system for the controlled analyses of gene function in Xenopus.