Achenbach Tatjana V, Brunner Bodo, Heermeier Kathrin
Aventis Pharma Deutschland GmbH, DI&A LG Functional Genomics, Industriepark Hoechst, Building G879, 65926 Frankfurt am Main, Germany.
Chembiochem. 2003 Oct 6;4(10):928-35. doi: 10.1002/cbic.200300708.
The postgenomic era is characterized by an almost intimidating amount of information regarding the sequences and expression of previously unknown genes. In response, researchers have developed an increasing interest in functional studies. At the start of such a study, one may have little more than sequence information and bioinformatic annotation. The next step is to hypothesize a potential role in the context of a cell. Testing of the hypothesis needs to be fast, cheap, and applicable to a large number of genes. Knockdown methods that rely on binding of antisense oligonucleotides to mRNA combined with a subsequent functional assay in cell culture fulfil these requirements: sequence information is sufficient for synthesis of active inhibitors. Depending on the in vitro model chosen, knockdown of gene expression can be achieved with medium or even high throughput. The two most popular methods of knockdown in cell culture are the use of antisense oligonucleotides that rely on ribonuclease H (RNAse H)-dependent cleavage of mRNA, and RNA interference triggered by small double-stranded RNA molecules. Both methods act in a sequence-specific manner and can give efficient knockdown. In both cases, researchers struggle with nonspecific "off-target" effects and the difficulty of site selection. Studies that compare the methods differ in their judgment as to which method is superior.
后基因组时代的特点是，关于以前未知基因的序列和表达的信息量几乎达到了令人望而生畏的程度。作为回应，研究人员对功能研究的兴趣与日俱增。在这类研究开始时，研究人员可能除了序列信息和生物信息学注释之外几乎一无所有。下一步是在细胞环境中假设一个潜在的作用。对这一假设的检验需要快速、廉价且适用于大量基因。依赖反义寡核苷酸与mRNA结合并随后在细胞培养中进行功能测定的敲低方法满足了这些要求：序列信息足以合成活性抑制剂。根据所选的体外模型，基因表达的敲低可以通过中等通量甚至高通量来实现。细胞培养中最常用的两种敲低方法是使用依赖核糖核酸酶H（RNAse H）切割mRNA的反义寡核苷酸，以及由小双链RNA分子触发的RNA干扰。这两种方法都以序列特异性方式起作用，并且可以实现有效的敲低。在这两种情况下，研究人员都面临非特异性“脱靶”效应和位点选择困难的问题。比较这两种方法的研究在判断哪种方法更优越方面存在差异。
