RNA interference by transgenesis ; a new approach for functional genomics

我们希望在小鼠中通过转基因（tg）表达引发RNA干扰。作为前奏，短干扰RNA（siRNA）被证明可以有效地降低生殖囊泡（GV）卵母细胞中靶mRNA的水平。我们选择了一系列目标，复制先前显示与一个丢失相关的表型，同时揭示其他新的表型。通过对单个GV卵母细胞的半定量逆转录酶PCR证实靶转录物消耗。接下来，我们验证了通过RNA聚合酶III指导发夹RNA（shRNA）表达的载体中的DNA指导的RNAi; shRNA靶点对应于酪氨酸酶、eGFP和瘦素，如我们最初的提议中所概述的。我们开发了一种新的基于荧光的共转染测定法，其中shRNA编码构建体与包含靶开放阅读框和随后的内部核糖体进入位点（ires）的报告基因共转染，以促进来自双顺反子mRNA的报告基因（venus）的表达。这种方法使我们能够证明，通过损失的干扰介导的荧光，目标转录水平显着降低组织培养，从而验证shRNA构建体之前，他们在转基因中的使用。这为在转基因小鼠品系的持续生产中使用载体铺平了道路。我们正在使用该测定系统来评估第二系列潜在的tgs，其表达由PolII指导。

We wished to elicit RNA interference by transgene(tg) expression in mice. As a prelude to this, short interfering RNAs(siRNAs) were shown effectively to reduce target mRNA levels in germinal vesicle(GV) oocytes. We selected a range of targets, reproducing phenotypes previously shown to be associated with the loss of one, whilst revealing novel phenotypes for others. Target transcript depletion was confirmed by semi-quantitative reverse transcriptase PCR on single GV oocytes. We next validated DNA-directed RNAi in vectors directing expression of hairpin RNAs(shRNAs) by RNA polymerase III ; shRNA targets corresponded to tyrosinase, eGFP and leptin as outlined in our original proposal. We developed a novel, fluorescence based co-transfection assay in which shRNA-encoding constructs were cotransfected with a reporter that comprised the target open reading frame followed by an internal ribosome entry site(ires) to facilitate expression of a reporter (venus) from a bicistronic mRNA. This approach allowed us to demonstrate through loss of reporter-meditated epifluorescence that target transcript levels were markedly reduced in tissue culture, thereby validating shRNA constructs prior to their use in transgenesis. This has paved the way for use of the vectors in the ongoing production of transgenic mouse lines. We are employing the assay system to evaluate a second series of potential tgs whose expression is directed by PolII.