Production of antisense RNA leads to effective and specific inhibition of gene expression in C. elegans muscle.
Production of antisense RNA leads to effective and specific inhibition of gene expression in C. elegans muscle.
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发表时间：
1991-10
期刊：
影响因子：
4.6
通讯作者：
A. Fire;D. Albertson;S. Harrison;D. Moerman
中科院分区：
文献类型：
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作者：
A. Fire;D. Albertson;S. Harrison;D. Moerman
我们已经使用反义策略有效地干扰了线虫体壁肌肉中存在的两个编码肌丝蛋白的基因的表达。UNC-22和UNC-54基因的DNA片段在旨在产生体壁肌肉RNA的载体中被放置在相反的方向上。当将产生的质粒注射到卵母细胞中时，会产生肌肉功能缺陷的后代。这些动物的表型与产生反义RNA的基因的功能降低和/或消除一致：UNC-22反义结构的肌丝抽动和解体，UNC-54反义结构的肌张力缺乏、运动缓慢和产蛋缺陷。一小部分受影响的动物会将肌肉缺陷的特征遗传给后代。在这些情况下，转化的DNA以高拷贝数存在，并与观察到的肌肉缺陷共分离。我们已经检测了几个UNC-22反义质粒转化系，以确定观察到的表型的机制基础。内源性UNC-22基因座的RNA产物在正常水平上存在，该RNA在与反义RNA同源的区域正确剪接。没有发现通过腺苷脱氨基修饰该RNA为肌苷的证据。在受影响的动物中，内源性UNC-22基因的蛋白质产物水平大大降低。检测到转化DNA产生的反义RNA比来自内源基因的正义RNA要丰富得多。这些数据表明，观察到的表型是由于干扰了基因表达的后期步骤，如运输到细胞质或翻译。
We have used an antisense strategy to effectively disrupt the expression of two genes encoding myofilament proteins present in C. elegans body wall muscles. DNA segments from the unc-22 and unc-54 genes have been placed in reverse orientation in vectors designed to produce RNA in body wall muscles. When the resulting plasmids are injected into oocytes, progeny with defects in muscle function are produced. These animals have phenotypes consistent with reduction and/or elimination of function of the gene to which antisense RNA has been produced: twitching and disorganization of muscle filaments for the unc-22 antisense constructs and lack of muscle tone, slow movement, and egg laying defects for the unc-54 antisense constructs. A fraction of the affected animals transmit the defective-muscle trait to subsequent generations. In these cases the transforming DNA is present at high copy number and cosegregates with the observed muscle defects. We have examined several of the unc-22 antisense plasmid transformed lines to determine the mechanistic basis for the observed phenotypes. The RNA product of the endogenous unc-22 locus is present at normal levels and this RNA is properly spliced in the region homologous to the antisense RNA. No evidence for modification of this RNA by deamination of adenosine to inosine was found. In affected animals the level of protein product from the endogenous unc-22 locus is greatly reduced. Antisense RNA produced from the transforming DNA was detected and was much more abundant than 'sense' RNA from the endogenous locus. These data suggest that the observed phenotypes result from interference with a late step in gene expression, such as transport into the cytoplasm or translation.