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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Silence of the genes
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Silence of the genes

机译：基因沉默

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The genomes of many species, including man, have been laid bare with hundreds of known and thousands of unknown genes. A major challenge in present day biology is to decipher the molecular function of the unknown genes. Gene knockout by homologous recombination has proven to be very useful but is laborious and expensive. Antisense and ribozyme technology have been useful but are not always reliable or robust. RNA interference has emerged as a novel pathway that offers great hope and promise to study the functions of a vast number of genes. The advent of this technology could not have come at a better time. Briefly, in invertebrates, long double-stranded RNA molecules are processed by the endonucle-ase dicer into 21- to 23-nt small interfering RNAs (siRNAs), which are then incorporated into RNA-induced silencing complex, a multicomponent nuclease complex that selects and degrades mRNAs that are homologous to the initially delivered double-stranded RNA (1, 2). In mammalian systems, introduction of long double-stranded RNA (>50 bp) results in systemic, nonspecific inhibition of translation due to activation of the PKR response. This formidable obstacle can be overcome by the use of synthetic siRNAs (<30 bp) that can be either delivered ex-ogenously (3) or expressed endogenously from RNA polymerase III promoters, resulting in a powerful tool for achieving specific down-regulation of target mRNAs (4-8). Currently, in mammalian systems, the only way to generate a whole-genome knockdown screening by using RNA interference is to systematically generate synthetic or polymerase III-transcribed siRNA hairpins for every known target gene. Because only 25% of selected target siRNA sequences are functional, several synthetic siRNAs need to be generated and tested for every target gene. Algorithms are being developed to predict effective siRNA sequences for efficient screening of large numbers of genes, but presently they have not been validated and may require automation (9).
机译：包括人类在内的许多物种的基因组已经暴露了数百个已知基因和数千个未知基因。当今生物学的主要挑战是破译未知基因的分子功能。已经证明通过同源重组的基因敲除非常有用，但是费力且昂贵。反义和核酶技术很有用，但并不总是可靠或稳健的。 RNA干扰已成为一种新颖的途径，为研究大量基因的功能提供了巨大希望和希望。这项技术的出现再合适不过了。简而言之，在无脊椎动物中，长双链RNA分子被核酸内切酶切酶加工成21至23 nt的小干扰RNA（siRNA），然后将其掺入RNA诱导的沉默复合物中，该复合物是一种多组分核酸酶复合物，可以选择并降解与最初递送的双链RNA（1、2）同源的mRNA。在哺乳动物系统中，由于激活PKR反应，引入长双链RNA（> 50 bp）会导致系统的非特异性翻译抑制。通过使用合成的siRNA（<30 bp）可以克服这一巨大的障碍，这些siRNA可以外源递送（3），也可以从RNA聚合酶III启动子内源表达，从而成为实现特异性下调靶标的强大工具mRNA（4-8）。当前，在哺乳动物系统中，通过使用RNA干扰进行全基因组敲除筛选的唯一方法是针对每个已知靶基因系统性地生成合成的或聚合酶III转录的siRNA发夹。由于只有25％的选定靶siRNA序列具有功能，因此需要生成多个合成siRNA并针对每个靶基因进行测试。正在开发算法来预测有效siRNA序列，以有效筛选大量基因，但目前尚未通过验证，可能需要自动化（9）。

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