Pol II–Expressed shRNA Knocks Down Sod2 Gene Expression and Causes Phenotypes of the Gene Knockout in Mice

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Abstract
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.