Pol II-expressed shRNA knocks down Sod2 gene expression and causes phenotypes of the gene knockout in mice

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.

Figure 1. shRNA Expression and Knockdown of Sod2 Gene Expression In Vivo

(A) Schematic illustration of the transgene construct. The shRNA was designed to mimic human miR-30a structure (for details, see [13]). (B) PCR analysis of tail DNA identified transgenic founders. C+ indicates positive control; C−, negative control. Numbers indicate examples of various transgenic lines. (C) Northern blots detected shRNA expression in transgene-positive line-8 mice, but not in line-60 mice. Total RNA (30 μg) was loaded in each lane. The tissues are lung (1), heart (2), skeletal muscle (3), kidney (4), liver (5), brain (6), stomach (7), and spleen (8). C+ is the siRNA-positive control. (D) Western blots of SOD2 protein compare the SOD2 levels in the above tissues between line-8 mice and wild-type mice. Due to different levels of SOD2 in different tissues, different amounts of total protein from different tissues had to be loaded in order to maintain the assay in linear range. The amounts of proteins are the following in micrograms: (1) 30; (2–4) 10; (5–6) 15; (7) 20; and (8) 40. + indicates transgene positive; −, transgene negative. (E) SOD2 mRNA levels in the above tissues from transgenic line-8 mice measured by real-time PCR (n = 4; all “n” indicates mouse numbers). The levels were normalized to the level of SOD2 mRNA in tissues from the wild-type littermates, which were set as 100% (column C). (F) Levels of SOD2 activity in tissue lysates of transgenic line-8 mice compared with the wild-type littermates (n = 4).

Figure 2. Consequence of the SOD2 Knockdown

(A) Histochemical staining reveals that SDH activity in the heart of transgenic line-8 mice was reduced compared with the wild-type littermates (B). (C) ROS levels are increased in fibroblasts from the skeletal muscles of transgenic line-26 (top panel) and line-8 (bottom panel) mice, compared with those from the wild-type mice (middle panel). AU, arbitrary units. (D) Fibroblasts from the transgenic line-8 mice have elevated sensitivity to oxidative stress compared with those from the wild-type mice, and this sensitivity can be corrected by expressing an RNAi-resistant SOD2 (line 8 + SOD2r). The data are means observed in cells isolated from four individual mice. Error bars are SEM. The asterisks indicate significant difference as compared to either WT or rescued cells (p < 0.05). (E) Western blot detects SOD2 protein levels in fibroblasts isolated from the skeletal muscle of the wild-type and transgenic line-8 mice. The third lane is from the line-8 cells transduced with RAd expressing the siRNA-resistant SOD2.

Figure 3. The Level of SOD2 Expression Was Knocked Down Further in Bigenic Transgenic Mice Generated by Crossing the Two Lines (Lines 8 and 26)

(A) Northern blots indicate that siRNA levels are further increased in the 8/26 bigenic mice. (B) Western blots demonstrate that SOD2 protein levels are further knocked down in the 8/26 bigenic mice. (C) Real-time PCR shows that SOD2 mRNA levels are further lowered in the 8/26 bigenic mice.

Figure 4. The 8/26 Bigenic Mice Display the Phenotype of a SOD2-Null Animal

(A) Retarded growth (7-d-old animals). (B) Dilated cardiomyopathy (H&E-stained coronal sections of heart). LV, left ventricle; RV, right ventricle. (C–F) Lipid deposition in heart (C and D) and liver (E and F) stained with Oil Red O.

Figure 5. In Vivo Expression of SOD2 shRNA with Authentic miR30a Structure Did Not Up-Regulate the Expression of OSA1 and STAT1

Levels of the mRNAs were determined by real-time PCR. The levels of OAS1 and STAT1 mRNAs in the shRNA transgenic tissues from line-8 mice were normalized to GAPDH mRNA and expressed relative to that of wild-type littermate tissues. Data represent means from four mice + SEM.