Chang Kenneth, Elledge Stephen J, Hannon Gregory J
Cold Spring Harbor Laboratory, Watson School of Biological Sciences, Howard Hughes Medical Institute, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA.
Nat Methods. 2006 Sep;3(9):707-14. doi: 10.1038/nmeth923.
Loss-of-function genetics has proven essential for interrogating the functions of genes and for probing their roles within the complex circuitry of biological pathways. In many systems, technologies allowing the use of such approaches were lacking before the discovery of RNA interference (RNAi). We have constructed first-generation short hairpin RNA (shRNA) libraries modeled after precursor microRNAs (miRNAs) and second-generation libraries modeled after primary miRNA transcripts (the Hannon-Elledge libraries). These libraries were arrayed, sequence-verified, and cover a substantial portion of all known and predicted genes in the human and mouse genomes. Comparison of first- and second-generation libraries indicates that RNAi triggers that enter the RNAi pathway through a more natural route yield more effective silencing. These large-scale resources are functionally versatile, as they can be used in transient and stable studies, and for constitutive or inducible silencing. Library cassettes can be easily shuttled into vectors that contain different promoters and/or that provide different modes of viral delivery.
功能丧失遗传学已被证明对于探究基因功能以及在生物途径的复杂回路中探究其作用至关重要。在许多系统中，在RNA干扰（RNAi）发现之前，缺乏允许使用此类方法的技术。我们构建了第一代短发夹RNA（shRNA）文库，其模仿前体微小RNA（miRNA），以及第二代文库，其模仿初级miRNA转录本（汉农 - 埃利奇文库）。这些文库经过排列、序列验证，覆盖了人类和小鼠基因组中所有已知和预测基因的很大一部分。第一代和第二代文库的比较表明，通过更自然的途径进入RNAi途径的RNAi触发物能产生更有效的沉默。这些大规模资源在功能上具有通用性，因为它们可用于瞬时和稳定研究，以及组成型或诱导型沉默。文库盒可以很容易地穿梭到含有不同启动子和/或提供不同病毒递送模式的载体中。
