RNA interference by transgenesis ; a new approach for functional genomics

我们希望通过转基因(TG)在小鼠中的表达来诱导RNA干扰。在此之前，短干扰RNA(SiRNAs)被证明能有效地降低生发泡(GV)卵母细胞中的靶mRNA水平。我们选择了一系列靶点，复制了以前被证明与失去一个相关的表型，同时揭示了其他表型的新表型。对单个GV卵母细胞进行半定量逆转录聚合酶链式反应，证实目的基因缺失。接下来，我们验证了DNA导向的RNAi载体通过RNA聚合酶III指导发夹RNA(ShRNAs)的表达；shRNA靶标对应于我们原始提案中概述的酪氨酸酶、绿色荧光蛋白和瘦素。我们开发了一种新颖的基于荧光的共转染实验，其中shRNA编码的构建体与包含靶向开放阅读框架的报告基因共转染，随后是内部核糖体进入位点(IRES)，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通过失去报道者思考的荧光来证明在组织培养中目标转录水平显著降低，从而在转基因使用之前验证了shRNA构建。这为将载体用于正在进行的转基因小鼠品系的生产铺平了道路。我们正在使用该检测系统来评估由PolII指导表达的第二系列潜在的TGS。

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.