Pol II-expressed shRNA knocks down Sod2 gene expression and causes phenotypes of the gene knockout in mice.
Pol II-expressed shRNA knocks down Sod2 gene expression and causes phenotypes of the gene knockout in mice.
复制标题
DOI：
10.1371/journal.pgen.0020010
复制
发表时间：
2006-01
期刊：
影响因子：
4.5
通讯作者：
Xu Z
中科院分区：
文献类型：
--
作者：
Xia XG;Zhou H;Samper E;Melov S;Xu Z
RNA干扰（RNAi）已越来越多地用于无脊椎动物和哺乳动物细胞的反向遗传学，并有可能成为哺乳动物基因敲除技术的替代方案。到目前为止，只有RNA聚合酶III（Pol III）表达的短发夹RNA（shRNA）已被用于制造表达shRNA的转基因小鼠。然而，广泛的敲除和诱导的基因敲除表型在出生后的小鼠尚未得到证实。先前的研究表明，Pol II合成微小RNA（miRNAs）-执行基因沉默功能的内源性shRNA。为了在哺乳动物中实现有效的基因敲除并产生基因敲除的表型，我们设计了一种构建体，其中Pol II（泛素C）启动子驱动具有模拟人miRNA miR-30 a结构的shRNA的表达。两个转基因株系显示广泛和持续的shRNA表达，并有效敲除靶基因Sod 2。这些小鼠存活，但具有SOD 2缺陷的表型。通过杂交这两个品系产生的双基因杂合小鼠显示出几乎检测不到的靶基因表达和与靶基因敲除一致的表型，包括生长缓慢、脂肪肝、扩张型心肌病和过早死亡。这种方法打开了RNAi的大门，广泛的一系列完善的Pol II转基因策略，并提供了一个技术上更简单，更便宜，更快的替代基因敲除同源重组在小鼠和其他哺乳动物物种的反向遗传学。反向遗传学通过改变基因并观察结果来研究基因功能。在哺乳动物中，反向遗传学的一种强有力的方法是通过同源重组进行基因敲除，其使基因突变以阻止其功能表达。利用这种方法，研究人员已经揭示了许多基因的功能。然而，这种方法相对复杂、耗时且成本高。此外，该方法仅限于小鼠研究，因为其在其他哺乳动物种属中尚未充分确立。这项研究的作者测试了一种使用RNA干扰（RNAi）的替代方法，这是真核生物中广泛保守的机制，可以介导基因特异性沉默。这些研究人员使用RNA聚合酶II（Pol II）表达短发夹RNA（shRNA），该短发夹RNA在转基因小鼠中触发编码Mn超氧化物歧化酶（SOD 2）的mRNA的破坏。这些小鼠表现出Sod 2敲除小鼠的典型表型，包括各种组织中氧化应激水平升高、肝脏和肌肉中脂肪沉积、扩张型心肌病和过早死亡。这些结果为RNAi打开了一扇大门，为广泛的Pol II转基因策略提供了一种技术上更简单，更便宜，更快的替代基因敲除的方法，用于小鼠和其他哺乳动物物种的反向遗传学。
RNA interference (RNAi) has been used increasingly for reverse genetics in invertebrates and mammalian cells, and has the potential to become an alternative to gene knockout technology in mammals. Thus far, only RNA polymerase III (Pol III)–expressed short hairpin RNA (shRNA) has been used to make shRNA-expressing transgenic mice. However, widespread knockdown and induction of phenotypes of gene knockout in postnatal mice have not been demonstrated. Previous studies have shown that Pol II synthesizes micro RNAs (miRNAs)—the endogenous shRNAs that carry out gene silencing function. To achieve efficient gene knockdown in mammals and to generate phenotypes of gene knockout, we designed a construct in which a Pol II (ubiquitin C) promoter drove the expression of an shRNA with a structure that mimics human miRNA miR-30a. Two transgenic lines showed widespread and sustained shRNA expression, and efficient knockdown of the target gene Sod2. These mice were viable but with phenotypes of SOD2 deficiency. Bigenic heterozygous mice generated by crossing these two lines showed nearly undetectable target gene expression and phenotypes consistent with the target gene knockout, including slow growth, fatty liver, dilated cardiomyopathy, and premature death. This approach opens the door of RNAi to a wide array of well-established Pol II transgenic strategies and offers a technically simpler, cheaper, and quicker alternative to gene knockout by homologous recombination for reverse genetics in mice and other mammalian species. Reverse genetics studies gene functions by altering a gene and observing the consequences. A powerful method of reverse genetics in mammals is gene knockout by homologous recombination, which mutates a gene to prevent its functional expression. Using this method, investigators have revealed the functions of many genes. However, this method is relatively complex, time-consuming, and costly. In addition, this method is limited to studies in mice because it is not well established in other mammalian species. The authors of this study tested an alternative method using RNA interference (RNAi), which is a widely conserved mechanism in eukaryotes and can mediate gene-specific silencing. These investigators used RNA polymerase II (Pol II) to express a short hairpin RNA (shRNA) that triggers destruction of the mRNA-encoding Mn superoxide dismutase (SOD2) in transgenic mice. These mice exhibit phenotypes that were typical in Sod2 knockout mice, including elevated levels of oxidative stress in various tissues, fat deposition in liver and muscles, dilated cardiomyopathy, and premature death. These results open the door of RNAi to a wide array of well-established Pol II transgenic strategies and offer a technically simpler, cheaper, and quicker alternative to gene knockout for reverse genetics in mice and other mammalian species.
登录
查看更多内容
影响因子：
4.5
作者：
Cai, XZ;Hagedorn, CH;Cullen, BR
通讯作者：
Cullen, BR
影响因子：
3.5
作者：
Hasuwa, H;Kaseda, K;Okabe, M
通讯作者：
Okabe, M
影响因子：
7.2
作者：
Gil, J;Esteban, M
通讯作者：
Esteban, M
影响因子：
4.5
作者：
Bracht, J;Hunter, S;Pasquinelli, AE
通讯作者：
Pasquinelli, AE
影响因子：
64.8
作者：
Denli, AM;Tops, BBJ;Hannon, GJ
通讯作者：
Hannon, GJ