XGX, HZ, and ZX conceived and designed the experiments. XGX, HZ, ES, and SM performed the experiments. XGX, HZ, ES, SM, and ZX analyzed the data. XGX, HZ, ES, and SM contributed reagents/materials/analysis tools. ZX wrote the paper.
XGX, HZ, and ZX are authors of a pending patent on the Pol II–shRNA construct used in this study.
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
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
Gene knockout by homologous recombination has been instrumental in investigating gene functions in mammals. It has been used to reveal gene functions in normal as well as in pathogenic pathways in vivo, and to generate models for many genetic disorders. However, the technical complexity, lengthy process, and high cost have limited its broad application. This is particularly problematic considering the fact that of the ~30,000 currently known mouse genes, only 10% have been knocked out, and even fewer are readily accessible by the research community [
RNAi is a widely conserved mechanism in eukaryotes [
In differentiated mammalian cells, long dsRNA activates RNA-dependent protein kinase PKR and type I interferon response, which leads to a nonspecific global translation depression and apoptosis [
The simplicity and specificity of RNAi has made RNAi a routine tool for investigation of gene functions in invertebrates and mammalian cells. Attempts have also been made to develop RNAi as an in vivo reverse genetics tool in mice. An early experiment directly injected long dsRNA into mouse embryonic stem cells. Successful knockdown and the phenotype of the gene deletion were observed in embryogenesis [
With the advent of Pol III promoter-directed synthesis of shRNAs, several groups engineered transgenic mice using Pol III–shRNA constructs [
In addition to Pol III, Pol II can also direct shRNA synthesis [
To test this idea, we used a construct that is composed of a human ubiquitin C promoter and an shRNA with the human miRNA miR-30a structure [
The construct UbC-SOD2hp-EGFP (
(A) Schematic illustration of the transgene construct. The shRNA was designed to mimic human miR-30a structure (for details, see [
(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 (
(F) Levels of SOD2 activity in tissue lysates of transgenic line-8 mice compared with the wild-type littermates (
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 [
To confirm the knockdown of
(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 (
(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.
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) 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.
(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.
Several studies have shown that some shRNA or siRNA could trigger interferon response [
Levels of the mRNAs were determined by real-time PCR. The levels of
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
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 [
The transgene construct that contains the hairpin targeting the
Mice were decapitated under anesthesia, and various tissues were quickly dissected, snap-frozen in liquid nitrogen, and stored at −80 °C. The total RNA was extracted from frozen mouse tissues using Trizol (Sigma, St Louis, Missouri, United States). Thirty micrograms of total RNA was fractionated on 15% polyacrylamide gels and transferred onto Hybond TM-N+ membrane (Amersham Biosciences, Little Chalfont, United Kingdom). After UV cross-linking, the membrane was probed with 32P-labeled synthetic RNA oligonucleotide complementary to the antisense strand of the mouse
The frozen mouse tissues were homogenized in ice-cold lysis buffer containing 0.4% NP-40, 0.2 mM Na3VO4, 20 mM HEPES (pH 7.9), and a cocktail of protease inhibitors (Complete-Mini; Sigma). The protein content in the cleared lysate was determined using the BCA assay. Equal amount of total proteins from transgenic and wild-type control animals was resolved by 15% SDS-PAGE and blotted onto GeneScreen Plus membrane (PerkinElmer, Wellesley, Massachusetts, United States). Proteins were detected using specific primary antibodies and the SuperSignal kit (Pierce Biotechnology, Rockford, Illinois, United States) and photographed using the Kodak Digital Image Station 440CF. The primary antibodies were: rabbit anti-Mn superoxide dismutase (SOD2; 1:1,000, Stressgen Biotechnologies, San Diego, California, United States) and mouse anti–glyceraldehye-3-phosphate dehydrogenase (GAPDH; 1:10,000; Abcam, Cambridge, United Kingdom). After detection of SOD2, the membrane was stripped for 30 min at 55 °C in a buffer containing 100 mM β-mercaptoethanol, 2% SDS (w/v), and 62.5 mM Tris-HCl (pH 6.7), and used again for detection of GAPDH immunoreactivity, which served as loading control.
Total RNA isolated using the TRI reagent was further purified with the RNeasy kit (Qiagen, Valencia, California, United States) and subjected to digestion on column with RNase-Free DNase (Qiagen). One microgram of purified total RNA from each sample was reversely transcribed to cDNA with oligo-dT primer using the RT kit (Invitrogen, Carlsbad, California, United States). The cDNA was used for quantitative PCR with SYBR green kit (Qiagen) according to manufacturer's instruction. The primer concentration was 500 nM. Cycling conditions were 15 min at 95 °C (to activate the hot-start Taq polymerase supplied with the SYBR Green detection kit), followed by 40 cycles of 15 s at 94 °C, 30 s at 60 °C, and 20 s at 72 °C. During amplification the fluorescence signal, which is proportional to the amount of dsDNA produced, was monitored. A complete amplification profile for each of the 96 wells of a PCR plate was obtained, which was used for the analysis. At the end of the PCR run, melting curves of the amplified products were obtained, which were used to determine the specificity of the amplification reaction. In pilot experiments, aliquots of the amplified products were separated on 3% agarose gels to ensure amplification of specific products of the predicted length. The amplification curves were used to calculate the threshold cycle number at which the amplification curve reaches the beginning of the linear phase of amplification. The threshold cycle number for
SOD2 activity was determined by inhibition of xanthine/xanthine oxidase–induced cytochrome C reduction [
Immediately following death, tissues were harvested and frozen on dry ice and sectioned at 20 μm thickness at −25 °C. SDH staining was performed and evaluated as previously described on frozen sections [
Fibroblasts were isolated from skeletal muscle in 15-d-old mice using the method modified from Crisona et al. [
ViraPower adenoviral kit (Invitrogen) was used. An RNAi-resistant
Oil Red O staining was performed on frozen sections. The fresh tissues collected as described above were frozen in powdered dry ice. The frozen sections (12 μm) were cut using a Cryostat and stained with Oil Red O solution on slides as follows: the sections were fixed in 10% formalin for 5 min, washed with several changes of phosphate-buffered saline, stained in prewarmed Oil Red O solution (60 °C; Sigma) for 8 min, washed again several times with distilled water, stained in Gill's hematoxylin solution for 30 s, and washed several times in distilled water. After mounted with coverslips, the stained sections were observed under microscope and photographed.
(A) Northern blots detect shRNA expression in transgene-positive line 26. 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).
(B) Western blots compare the SOD2 protein levels in the above tissues between line-26 mice and wild-type mice. + indicates transgene positive, and − indicates transgene negative. The amounts of proteins are loaded in the same order as described in
(C) SOD2 mRNA levels in the above tissues from transgenic line 26 measured by real-time PCR (
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(A) Testing the specificity of the primers used for real-time PCR. HEK293 cells were used as reference for gene copy number of ubiquitin C. NT, nontransgenic.
(B) Estimation of UbC-SOD2hp-EGFP gene copy numbers. A 118-bp segment in human ubiquitin C promoter was amplified using a pair of specific primers. Also amplified was human and mouse
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(A) Drosha was knocked down by RNAi in the fibroblasts from wild-type and line-8 transgenic mice. The cells were transduced with adenoviral vectors expressing an shRNA against mouse Drosha (shRNA stem sequence: 5′-GGATGAAGATTTAGAGAGTTC-3′). Four days after transduction, the total RNA was extracted from the cells and used for RT-PCR to detect Drosha. The ribosomal RNA L17 was magnified in parallel as input control. The PCR was run for 28 cycles using the following primers for Drosha: 5′-GAGCCTAGAGGAAGCCAAACA-3′ (forward) and 5′-GCCGGACGTGAGTGAAGAT-3′ (reverse); for L17: 5′-CGGTATAATGGTGGAGTTG-3′ (forward) and 5′-ACCCTTAAGTTCAGCGTTACT-3′ (reverse).
(B) No EGFP fluorescence could be detected in fibroblasts isolated from line-8 transgenic mice.
(C) The same field as in (B) was stained with DAPI.
(D) Three days after the fibroblasts were transduced with an adenoviral vector that expressed an shRNA against mouse Drosha, EGFP fluorescence was detected.
(E) The same field as in (D) was stained with DAPI.
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We thank members of the Xu lab and Dr. Alonzo Ross for advice and support, Phillip Zamore for discussion and commenting on the manuscript, and Steve Jones and the University of Massachusetts Medical School transgenic core for pronuclear injection. This work was supported by grants from the Amyotrophic Lateral Sclerosis (ALS) Association, National Institutes of Health (NIH)/National Institute of Neurological Disorders and Stroke (NINDS) (R01NS048145), NIH/National Institute on Aging (NIA) (R21AG023808), and The Robert Pachard Center for ALS Research at Johns Hopkins to ZX, and NIH/NIA (RO1AG18679) to SM. The contents of this report are solely the responsibility of the authors and do not necessarily represent the official views of the NIH.
double-stranded RNA
enhanced green fluorescent protein
micro RNA
RNA polymerase
RNA-induced silencing complex
RNA interference
succinate dehydrogenase
short hairpin RNA
small interfering RNA