Giovannini Natalia, Rungger Duri
Station de Zoologie Expérimentale, Department of Animal Biology, University of Geneva, CH-1224 Chêne Bougeries, Switzerland.
Dev Growth Differ. 2002 Apr;44(2):147-59. doi: 10.1046/j.1440-169x.2002.00630.x.
Expression of the Xbrachyury (Xbra) gene was inhibited by antisense RNA synthesized in situ from an expression vector read by RNA polymerase III, injected into the fertilized egg or the 2-cell stage embryo of Xenopus laevis. Antisense-treated embryos had markedly reduced levels of Xbra mRNA and protein, and showed deficiencies in mesodermal derivatives and axis formation. In particular, organization of the posterior axis was affected, but often the anterior axis was also reduced. Some embryos failed to form mesoderm altogether and remained amorphous. The antisense effect is dose-dependent and may be "rescued" by overexpression of Xbra. In Xbra-deficient embryos, expression of several mesodermal genes (Xvent, pintallavis, Xlim, Xwnt-8 and noggin) was reduced to varying degrees, whereas goosecoid levels remained normal. The modified expression levels were partly normalized when Xbra deficiency was rescued. The observation that antisense inhibition yields slightly different phenotypes from dominant-negative inhibition suggests the recommendation of using several surrogate genetic approaches to determine the functional role of a gene in Xenopus development.
通过RNA聚合酶III读取的表达载体原位合成反义RNA，将其注射到非洲爪蟾的受精卵或二细胞期胚胎中，从而抑制Xbrachyury（Xbra）基因的表达。经反义处理的胚胎中Xbra mRNA和蛋白质水平显著降低，并表现出中胚层衍生物和轴形成方面的缺陷。特别是，后轴的组织受到影响，但前轴通常也会缩短。一些胚胎完全未能形成中胚层，仍呈无定形。反义效应具有剂量依赖性，并且可以通过Xbra的过表达“挽救”。在Xbra缺陷型胚胎中，几个中胚层基因（Xvent、pintallavis、Xlim、Xwnt - 8和noggin）的表达不同程度降低，而goosecoid水平保持正常。当Xbra缺陷得到挽救时，改变的表达水平部分恢复正常。反义抑制产生的表型与显性负抑制略有不同，这一观察结果表明建议使用几种替代遗传方法来确定基因在非洲爪蟾发育中的功能作用。
