The construct UbC-SOD2hp-EGFP (; also see [13
]) consists of the human ubiquitin C promoter, an shRNA-coding hairpin placed in the first intron, followed by the enhanced green fluorescent protein (EGFP) coding sequence and a poly-adenylation signal. The shRNA-coding hairpin mimics human microRNA miR-30a structure and target mouse Sod2
mRNA. Using this construct, we generated transgenic mice by pronuclear injection of fertilized eggs. Screening of 62 founders yielded five positive lines (A). Northern blots showed that two of the five lines (lines 8 and 26) expressed siRNA broadly (B, C, and S1
A) and this expression was stable over multiple generations and during aging (the same level of expression was observed in animals from 40 to 200 d old; unpublished data). By real-time PCR line 8 carried a single copy of the transgene while line 26 carried three copies (Figure S2
). The transgene copy number did not predict the level of siRNA levels since line 8 had a higher level of siRNA than line 26 (see below).
shRNA Expression and Knockdown of Sod2 Gene Expression In Vivo
The pattern of expression in transgenic mice differed from the pattern in cultured cells in two regards: none of these transgenic lines expressed detectable EGFP and shRNA (only siRNA was detected). This contrasts with what we observed in cultured cells, in which both were detectable [13
]. Nevertheless, the siRNA knocked down the abundance of SOD2 as indicated by the decreased levels of the protein (D and S1
B), mRNA (E and S1
C), and enzyme activity (F). The rapid processing of pre-miRNA in vivo probably caused the lack of shRNA detection. This explanation is consistent with the lack of pre-miRNA detection for other endogenous miRNAs in mammalian cells [34
]. The lack of EGFP was puzzling. One possibility was that the transgene was altered. This was ruled out by sequencing the transgene extracted by PCR, which revealed no alteration in the transgene structure. Another possibility was that the pri-miRNA processing in vivo was highly efficient, so that the processing of pri-miRNA occurred before the splicing, and, consequently, the mRNA could not be properly spliced and exported to the cytoplasm for EGFP expression. The processing of pri-miRNA involves Drosha [14
]. Therefore, if our hypothesis was correct, inhibition of Drosha expression should lead to EGFP expression. To test this, we transduced fibroblasts isolated from the skeletal muscle of the transgenic mice using a recombinant adenovirus that expresses an shRNA against Drosha. By RT-PCR the Drosha mRNA levels were substantially reduced (Figure S3
A), indicating the effectiveness of the shRNA. While the nontransduced cells showed no detectable EGFP fluorescence (Figure S3
B and S3
C), the transduced cells expressed EGFP (Figure S3
D and S3
E). This result supports our hypothesis.
To confirm the knockdown of Sod2
gene expression, we examined the in vivo consequence of SOD2 deficiency. First, we compared the activity of the mitochondrial enzyme succinate dehydrogenase (SDH) between the transgenic and wild-type animals, because SOD2 knockout is known to cause a decrease in SDH activity [37
]. We stained tissue sections from the heart using histochemical staining, and observed a decrease in the staining intensity in line 8 (A and B). Second, we isolated fibroblasts from skeletal muscle and measured the levels of superoxide in these cells. We observed that the superoxide levels were increased in the two transgenic lines (C). This increase was higher in line 8 than in line 26, and, therefore, was correlated with the degree of SOD2 knockdown (see below). Third, we tested the sensitivity of the fibroblasts to oxidative stress induced by t-butylhydroperoxide (t-BuOOH) treatment. Fibroblasts from transgenic line 8 showed a higher sensitivity than the wild-type cells (D), consistent with the lowered levels of SOD2 protein in these cells (E). To ensure that the enhanced sensitivity was caused by the knockdown of SOD2 expression, we transduced the fibroblasts with a recombinant adenovirus that expressed a Sod2
gene whose mRNA was resistant to the siRNA expressed by the transgene. Expression of this SOD2 molecule (E) rescued the cells from their hypersensitivity to oxidative stress (D).
Consequence of the SOD2 Knockdown
Based on these data, we conclude that ubiquitin C promoter–directed shRNA synthesis effectively silenced the target molecule in transgenic mice. Despite this significant knockdown (by 60%–90%) in all the tissues examined and the evidence of functional SOD2 deficiency, the two transgenic lines were viable to 400 d (observed to date). SOD2-null phenotypes, including small body size, dilated cardiomyopathy, lipid deposition in liver and heart, and premature death, were not observed. To determine whether knockout phenotypes could be generated, we crossed the two lines that expressed the siRNA to generate bigenic heterozygous transgenic mice. We took this approach because it was advantageous compared with generating homozygous animals of each lines, the phenotype of which could be complicated by the potential gene disruption at the transgene insertion site.
The line 8/26 bigenic mice expressed a higher level of siRNA than either of the singly transgenic lines (A), leading to knockdown of SOD2 protein and mRNA to nearly undetectable levels (B and C). These mice exhibited phenotypes similar to the previously reported SOD2 knockout mice [35
], including smaller body size than the wild-type littermates (A) and death within 20 d after birth (in 34 bigenic animals that we obtained so far). In addition, they developed dilated cardiomyopathy (B) and had increased lipid deposition in the heart (C and D) and the liver (E and F).
The Level of SOD2 Expression Was Knocked Down Further in Bigenic Transgenic Mice Generated by Crossing the Two Lines (Lines 8 and 26)
The 8/26 Bigenic Mice Display the Phenotype of a SOD2-Null Animal
Several studies have shown that some shRNA or siRNA could trigger interferon response [39
]. This raises the possibility that interferon response might be responsible for the phenotype observed in these mice. To test this, we examined the levels of two genes known to be dramatically induced by the dsRNA-triggered interferon response, 2′,5′-oligoadenylate synthetase 1 (OSA1),
and signal transducer and activator of transcription 1 (STAT1)
]. Real-time PCR analysis of the mRNA of OSA1
failed to detect changes in the expression levels of these two genes in the heart, spleen, and liver in the transgenic line 8 (), indicating no interferon response in these transgenic mice.
In Vivo Expression of SOD2 shRNA with Authentic miR30a Structure Did Not Up-Regulate the Expression of OSA1 and STAT1
Taken together, the phenotypes are likely caused by the specific effect of SOD2 knockdown because (1) the siRNA was expressed widely; (2) the consequences of SOD2 deficiency were observed in the transgenic mice; (3) in cells isolated from these mice the hypersensitivity to oxidative stress was corrected by the Sod2 gene that was resistant to the siRNA; (4) the phenotype typical of the SOD2-null mouse was observed when siRNA levels were increased in the bigenic 8/26 mice; and (5) levels of OSA1 and STAT1, two molecules involved in the dsRNA-induced interferon response, were unchanged. These results demonstrate that Pol II–mediated expression of shRNA in transgenic mice can be used to investigate gene functions in mammals.
Thus, the Pol II–directed synthesis of shRNA can be an alternative to gene knockout technology for reverse genetics in mammals. Although gene knockout remains a useful approach for the complete gene deletion or gene modification, our RNAi approach can achieve near knockout conditions and is economical in cost and time. In addition, the construct design is simple and in principle not different from the standard transgene design for gene overexpression. The placement of the shRNA-encoding hairpin is flexible: it can be placed in introns or in the 3′ untranslated regions [13
], and the shRNA-encoding transcript is not required to encode a protein [13
]. Therefore, a wide variety of transgene promoters that have already been successfully used to overexpress genes in transgenic mice can be readily adapted for suppressing the genes. When spatially and temporally specific promoters are used, controlled suppression of the target gene can be achieved. Furthermore, this approach provides a more flexible system to model hypomorphic allele function. While the standard knockout technology reduces the target gene expression to 50% or 0% in most instances, transgenic RNAi can reduce the target gene to more variable degrees in different lines of transgenic mice or by induction using small molecules in transgenic mice made with inducible Pol II promoters [43
]. The simplicity of this approach can accelerate the generation of models for diseases that are caused by various degrees of genetic hypomorphism, such as autosomal dominant polycystic kidney disease [45
], various cancers [46
], and other diseases. Finally, gene knockout by homologous recombination has not been established in other mammalian species. Current Pol III strategies rely on the crosses with numerous Cre-expression transgenic mouse lines [24
], which are not available in other mammalian species. Our knockdown strategy can overcome these limitations and be used to carry out reverse genetics and to generate disease models in other mammalian species.